Dysfunctional antigen-specific cd8+ t cells in the tumor microenvironment

ABSTRACT

Provided herein are compositions and methods for detecting and/or targeting dysfunctional tumor antigen-specific CD8 +  T cells in the tumor microenvironment for diagnostic, therapeutic and/or research applications. In particular, dysfunctional tumor antigen-specific CD8 +  T cells are detected and/or targeted via their expression of cell surface receptors described herein, such as 4-1BB, LAG-3, or additional markers that correlate with 4-1BB and LAG-3 expression, such as markers differentially expressed on the surface of the T cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser. No. 16/476,219, filed Jul. 5, 2019, now allowed, which is a § 371 National Entry of PCT/US18/14008, filed Jan. 17, 2018, which claims priority to U.S. Provisional Patent Application Ser. No. 62/447,199, filed Jan. 17, 2017, which is incorporated by reference in its entirety.

SEQUENCE LISTING

The text of the computer readable sequence listing filed herewith, titled “35120-303_SEQUENCE_LISTING”, created Jan. 6, 2023, having a file size of 53,909 bytes, is hereby incorporated by reference in its entirety.

FIELD

Provided herein are compositions and methods for detecting and/or targeting dysfunctional tumor antigen-specific CD8⁺ T cells in the tumor microenvironment for diagnostic, therapeutic and/or research applications. In particular, dysfunctional tumor antigen-specific CD8⁺ T cells are detected and/or targeted via their expression of cell surface receptors described herein, such as 4-1BB, LAG-3, or additional markers that correlate with 4-1BB and LAG-3 expression, such as markers differentially expressed on the surface of the T cells.

BACKGROUND

The immune system plays a critical role in protecting the host from cancer (Vesely et al., 2011; incorporated by reference in its entirety). Innate sensing of tumors leads to an adaptive T cell response through the presentation of tumor-associated antigens (TAAs) derived from mutations and epigenetic changes that contribute to carcinogenesis (Gajewski et al., 2013; incorporated by reference in its entirety). Spontaneously-primed CD8+ T cells home to tumor sites in mouse tumor models (Harlin et al., 2009; Fuertes et al., 2011; incorporated by reference in their entireties) and in a subset of patients with advanced cancer (Harlin et al., 2006; incorporated by reference in its entirety). These tumor-infiltrating lymphocytes (TIL) have the ability to recognize tumor antigens and are believed to contribute to tumor control in cancer patients, based on the correlation between activated CD8+ T cell infiltration with improved prognosis and response to immunotherapy (Fridman et al., 2012; Tumeh et al., 2014; incorporated by reference in their entireties). However, without additional manipulation, this endogenous anti-tumor response is usually not sufficient to mediate complete rejection of an established tumor (Gajewski, 2007b; Pardoll, 2012; Baitsch et al., 2011; Gajewski et al., 2006; Larkin et al., 2015). Data accumulated over the past several years have indicated that tumors with spontaneous anti-tumor T cell responses have high expression of immune-inhibitory pathways that subvert the effector phase of the response. These include PD-L1/PD-1 interactions (Pardoll, 2012; incorporated by reference in its entirety), recruitment of CD4+Foxp3+ regulatory T (Treg) cells (Gajewski, 2007a; incorporated by reference in its entirety), and metabolic dysregulation by indoleamine-2,3-dioxygenase (IDO) (Spranger et al., 2013; incorporated by reference in its entirety). However, even when CD8+ T cells specific for tumor antigens are isolated from tumors, away from these extrinsic immune inhibitory factors, they still show altered functional properties ex vivo (Harlin et al., 2006; Baitsch et al., 2011; incorporated by reference in their entireties).

Expression of PD-1 has been described to identify tumor-specific exhausted T cells (Ahmadzadeh et al., 2009; Fourcade et al., 2012; Wu et al., 2014; Gros et al., 2014; incorporated by reference in their entireties). However, T cells expressing PD-1 in the context of chronic infection can still retain effector function (Wherry and Kurachi, 2015; incorporated by reference in its entirety), and PD-1 is not required for the induction of T cell exhaustion (Odorizzi et al., 2015; incorporated by reference in its entirety). In addition to PD-1, several additional co-inhibitory receptors, including CD223 (LAG-3), CD244 (2B4), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), hepatitis A virus cellular receptor 2 (TIM-3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), are also be expressed on dysfunctional T cells and expression of a greater number of inhibitory receptors has been correlated with diminished cytokine secretion (in particular IFN-g and TNF-a) as well as proliferative capacity (Blackburn et al., 2009; incorporated by reference in its entirety). Expression of these receptors has been observed in both viral and cancer models, however, a complete analysis of both co-inhibitory and co-stimulatory receptors on the same population is lacking in the tumor setting.

SUMMARY

Provided herein are compositions and methods for detecting and/or targeting dysfunctional tumor antigen-specific CD8⁺ T cells in the tumor microenvironment for diagnostic, therapeutic and/or research applications. In particular, dysfunctional tumor antigen-specific CD8⁺ T cells are detected and/or targeted via their expression of cell surface receptors described herein, such as 4-1BB, LAG-3, or additional markers that correlate with 4-1BB and LAG-3 expression, such as markers differentially expressed on the surface of the T cells (e.g., PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM and Sema7a).

In some embodiments, provided herein are methods of treating a subject with cancer comprising administering an agent that specifically targets dysfunctional tumor antigen-specific CD8⁺ T cells. In some embodiments, the subject suffers from a solid tumor cancer. In some embodiments, the tumor allows T cell infiltration, but is resistant to immunotherapies. In some embodiments, the tumor environment comprises dysfunctional tumor antigen-specific CD8⁺ T cells. In some embodiments, contacting the dysfunctional tumor antigen-specific CD8⁺ T cells with an anti-4-1BB and/or anti-LAG3 agent. In some embodiments, the anti-4-1BB and/or anti-LAG3 agent is an antibody, antibody fragment, or antibody mimetic molecule. In some embodiments, methods further comprise co-administration of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic or an immunotherapeutic agent. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent selected from the list consisting of cell-based therapies, monoclonal antibody (mAb) therapy, cytokine therapy, and adjuvant treatment. In some embodiments, the immunotherapeutic agent is a mAb therapy selected from the list consisting of anti-CTLA-4 monoclonal antibodies and/or anti-PD-L1 monoclonal antibodies. In some embodiments, the immunotherapeutic agent is a cell-based therapy selected from the list consisting of dendritic-cell therapy and T-cell therapy. In some embodiments, the additional therapeutic agent targets one of the markers/receptors listed in Table 2. In some embodiments, the additional therapeutic targets a marker/receptor expressed on the surface of the T cells. In some embodiments, the additional therapeutic targets PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM or Sema7a. In some embodiments, the additional therapeutic agent targets Nrn1, Sema7a, or CRTAM.

In some embodiments, provided herein are methods of treating a subject with cancer comprising administering a therapeutic agent that specifically targets dysfunctional tumor antigen-specific CD8⁺ T cells, wherein the agent targets one of the receptors listed in Table 2. In some embodiments, the therapeutic targets a marker/receptor expressed on the surface of the T cells. In some embodiments, the therapeutic targets PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM or Sema7a. In some embodiments, the therapeutic agent targets Nrn1, Sema7a, or CRTAM. In some embodiments, the therapeutic agent is an antibody, antibody fragment, or antibody mimetic molecule that binds the target marker/receptor. In some embodiments, the therapeutic agent is an anti-Nrn antibody, antibody fragment, or antibody mimetic molecule. In some embodiments, the therapeutic agent is an anti-Sema7a antibody, antibody fragment, or antibody mimetic molecule. In some embodiments, the therapeutic agent is an anti-CRTAM antibody, antibody fragment, or antibody mimetic molecule.

In some embodiments, provided herein are compositions comprising: (a) one or more of an anti-4-1BB agent, an anti-LAG-3 agent, an anti-Nrn1 agent, an anti-Sema7a agent, and an anti-CRTAM agent; and (b) an immunotherapeutic agent, said composition formulated for therapeutic delivery to a subject. In some embodiments, the anti-4-1BB agent, anti-LAG-3 agent, anti-Nrn1 agent, anti-Sema7a agent, and/or anti-CRTAM agent is an antibody, antibody fragment, or antibody mimetic molecule.

In some embodiments, provided herein are compositions comprising: (a) an agent that targets and/or binds one of PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A; and (b) an immunotherapeutic agent, said composition formulated for therapeutic delivery to a subject.

In some embodiments, provided herein are methods comprising: (a) testing CD8⁺ T cells from a cell population to determine whether they co-express LAG-3 and 4-1BB; and (b) administering one or more agents that target and/or bind one of PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A. In some embodiments, the agent is an anti-Nrn1 agent, an anti-Sema7a agent, and an anti-CRTAM agent. In some embodiments, the anti-Nrn1 agent, anti-Sema7a agent, and/or anti-CRTAM agent is an antibody, antibody fragment, or antibody mimetic molecule. In some embodiments, testing is performed in vitro.

In some embodiments, provided herein are methods of identifying dysfunctional T cells by testing said cells for co-expression of 4-1BB and LAG-3. In some embodiments, provided herein are methods of identifying dysfunctional T cells by testing said cells for expression of one or more of the markers/receptors of Table 2 (e.g., a T-cell surface marker/receptor (e.g., PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, TMEM126A).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-J. Co-expression of 4-1BB and LAG-3 identifies a significant fraction of the CD8⁺ TIL compartment found in progressing tumors. (A) Representative analysis of 4-1BB and LAG-3 expression on CD8⁺ T cells from B16. SIY tumors and the spleen and TdLN from tumor bearing mice on day 7, 14 and 21 after s.c. tumor inoculation. (B-D) Longitudinal summary of the composition, n=5; four to five independent experiments per time point, (C) absolute cell number, n=5; seven to nine independent experiments per time point, and (D) cellular density of the CD8⁺4-1BB/LAG-3 TIL subpopulations, n=5; two to five independent experiments per time point. Absolute cell numbers were determined by acquiring the complete tumor sample by flow cytometery. (E) Day 14 summary of the proportion of the CD8⁺4-1BB/LAG-3 TIL subpopulations that are Ki67⁺. n=3-5; two independent experiments. (F) Summary of BrdU uptake on day 13 in the CD8⁺4-1BB/LAG-3 TIL subpopulations after a 24 hour BrdU pulse. n=5; three independent experiments. (G-I) Representative flow plots (G and H) and summary (I) of the 4-1BB/LAG-3 populations in other tumor models. Mice were inoculated with 2×10⁶ C1498. SIY, MC38. SIY, EL4. SIY, B16 Parental, MC57. SIY or 1969. SIY subcutaneously and analyzed for 4-1BB and LAG-3 expression on day 14 after tumor inoculation. n=3-5; two to 5 independent experiments for each time point. (J) Mice were inoculated on both flanks with 2×10⁶ MC57. SIY or B16. SIY, at indicated time points tumors from each mouse were pooled and analyzed for co-expression of 4-1BB and LAG-3 in the CD8⁺ TIL compartment. n=3-5; two independent experiments for each time point. All error bars indicate ±SEM. *:P<0.05, **:P<0.01, ***:P<0.001. A two-way ANOVA with Bonferroni post-hoc test was used for (B, C, D, H) longitudinal studies and Kruskal-Wallis (non-parametric) test was used for (E and F) analysis at one time-point.

FIG. 2A-G. Egr2 and a component of the Egr2-transcriptional network are enriched in 4-1BB⁺LAG-3⁺ CD8⁺ TILs. (A) Representative flow plot and summary of Egr2^(EGFP) expression. Egr2^(EGFP) mice were inoculated with 2×10⁶ B16. SIY tumors s.c. CD8⁺ T cells from the tumor, TdLN and spleen were analyzed for Egr2^(EGFP) expression on day 7 and day 14. n=4-5; two-independent experiments. (B) Expression of Egr2 target genes (Zheng et al., 2013). CD8⁺ TILs from day 14 tumor bearing mice were sorted based on high or low expression of Egr2^(EGFP) and analyzed directly for expression of Egr2 targets by qRT-PCR. Two tumors on opposite flanks pooled per mouse. n=3; two independent experiments. (C) Representative flow plots and summary of the 4-1BB/LAG-3 subpopulations in CD8⁺ Egr2GFP^(hi) and Egr2GFP^(lo) TILs on day 7 and 14. n=4-5. Two-independent experiments per time point. (D) Expression of Egr2 targets in the 4-1BB⁺LAG-3⁺ and 4-1BB⁻LAG-3 subpopulations. The subpopulations were sorted and analyzed directly for the expression of targets by qRT-PCR. Two tumors on opposite flanks pooled per mouse. n=4; two-independent experiments. (E) Egr2^(flox/flox)×pLCKCreERT2×YFP-Rosa26 mice given 5 doses of tamoxifen by gavage and inoculated 3 days later with 2×10⁶ B16. SIY cells. YFP⁺ or YFP⁻ CD8⁺ TILs were sorted and analyzed for Egr2 transcript directly and after in vitro stimulation. Two tumors on opposite flanks pooled per mouse. n=3; two independent experiments. (F) Representative flow plots and summary of 4-1BB/LAG-3 co-expression in YFP⁺ or YFP⁻ CD8⁺ TILs on day 7 and 14. n=3; two independent experiments. (G) Expression of Egr3 and Hif1α in Egr2GFP^(hi) and Egr2GFP^(lo) from day 7 CD8⁺ TILs isolated from Egr2GFP mice. n=5; two-independent experiments. Error bars indicate ±SEM. *:P<0.05, **:P<0.01, ***:P<0.001. A two-way ANOVA with Bonferroni post-hoc test was used for longitudinal studies (A and C) and a Mann-Whitney test was used to compute significance in (B, D, E, F and G).

FIG. 3A-H. Co-expression of 4-1BB and LAG-3 identifies tumor antigen-specific TILs in progressing tumors. (A) Representative CDR3β distributions from the different 4-1BB/LAG-3 subpopulations and CD8⁺ T cells isolated from the spleen. Boxed regions represent dominant peaks in the 4-1BB⁺LAG-3⁺ CD8⁺ TIL subpopulation. (B) As a measure of skewness, the Hamming Distance (HD) for each Vβ spectratype was calculated between each TIL subpopulation and CD8⁺ T cell spleen population within the same mouse. As a control the HDs from CD8⁺ splenocyte populations between mice (grey bar) were calculated. n=3; one independent experiment. (C-D) Representative flow analysis of the 4-1BB/LAG-3 subpopulation in H-2K^(b)/SIY⁺ and H-2K^(b)/SIY⁻ CD8⁺ TILs on day 14 after B16. SIY and MC38. SIY or (D) MC57. SIY and 1969. SIY tumor inoculation. n=3-4; three to five independent experiments. (E) Summary of the composition of H-2K^(b)/SIY⁺ and H-2K^(b)/SIY⁻ CD8⁺ TILs co-expressing 4-1BB and LAG-3 comparing B16. SIY, MC38. SIY, MC57. SIY and 1969. SIY tumors on day 14 after tumor inoculation. n=5; three to four independent experiments. (F-H) On day 7 after tumor inoculation 1×10⁶ P14/CD45.2 and 2C/CD45.1/2 Tg T cells were adoptively transferred, via tail vein, into CD45.1 congenic tumor bearing hosts and analyzed for the (F) total number of recovered cells in the tumor, (G and H) profile of 4-1BB and LAG-3 expression in 2C, P14 and host CD8⁺ TILs. n=5; two-independent experiments. All error bars indicate ±SEM. *:P<0.05, **:P<0.01, ***:P<0.001. A Kruskal-Wallis (non-parameteric) test was used for (B) spectratype analysis and (E and F) H-2K^(b)/SIY analysis. A two-way ANOVA with Bonferroni post-hoc test was used for (H) 2C, Host and P14 composition analysis.

FIG. 4A-G. Co-expression of 4-1BB and LAG-3 but not PD-1 define dysfunctional CD8⁺ TILs with diminished IL-2. (A and B) Sorted cells from day 14 B16. SIY tumor bearing mice were stimulated in vitro with anti-CD3F and anti-CD28 for 12 hours and analyzed for (A) Il-2 transcript by qRT-PCR and (B) IL-2 protein by ELISA. Two tumors on opposite flanks pooled per mouse. n=4-5; three independent experiments. (C) Egr2GFP^(hi) and Egr2GFP^(lo) TILs were sorted from day 14 B16. SIY tumor bearing Egr2^(GFP) mice and stimulated in vitro for 12 hours and analyzed for Il-2 transcript by qRT-PCR. Two tumors on opposite flanks pooled per mouse. n=5; two independent experiments. (D) On day 7 after tumor inoculation 1×10⁶ 2C/CD45.1/2 Tg T cells were transferred into mice, 7 days later host 4-1BB⁺LAG-3⁺ T cells sorted from the tumor and 2C T cells sorted from the tumor or TdLN were stimulated in vitro and analyzed for expression of Il-2 transcript by qRT-PCR. Two tumors on opposite flanks pooled per mouse. n=3; two independent experiments. (E and F) Representative flow analysis of PD-1 expression on 4-1BB/LAG-3 CD8⁺ TIL subpopulations and (F) summary of the composition of the 4-1BB⁻LAG-3⁻PD-1⁺ subpopulation in the CD8⁺ TIL compartment on day 14 and 21. n=5; three independent experiments. (G) 4-1BB-LAG-3-PD-1+ and LAG-3⁺4-1BB⁺ CD8⁺ TILs were sorted from day 14 tumor bearing mice, stimulated in vitro and analyzed for Il-2 transcript by qRT-PCR. Two tumors on opposite flanks pooled per mouse. n=3; two independent experiments. All error bars indicate ±SEM. *:P<0.05, **:P<0.01, ***:P<0.001 ****:P<0.0001. A Kruskal-Wallis (non-parametric) test was used for analysis of multiple comparisons (A, B, and D) and a Mann-Whitney test was used for pair-wise comparisons (C and G).

FIG. 5A-E. Dysfunctional CD8⁺ TILs retain IFN-7 production, cytolytic capacity and produce Treg-recruiting chemokines. (A) Longitudinal analysis CD8⁺ TIL subpopulation cytokine production capacity. CD8⁺ TIL subpopulations were sorted and stimulated with anti-CD3F and anti-CD28 for 10-12 hours and the concentration of IL-2, IFN-γ and TNF-α was measured. Concentration was normalized to cell number. Two tumors on opposite flanks pooled for day 7 and 14. n=4-5; two-independent experiments. (B) Ifn-γ Tnf-α and Gzmb transcript levels in the 4-1BB/LAG-3 subpopulations analyzed directly ex vivo. Two tumors on opposite flanks pooled per mouse. n=3-5; three-independent experiments. (C) Representative flow plot and summary of IFN-γ production analyzed directly ex vivo. Briefly, 100 μl of PBS containing 2 mg/mL GolgiPlug was injected intratumorally on day 14 after tumor inoculation. 8 hours later TILs were isolated. All steps were performed on ice with media containing 1 mg/mL GolgiStop until fixation. n=5; two independent experiments. (D) CD8⁺ TIL subpopulations at indicated time points were sorted and plated with 50,000 P815 target cells and 1 μg/mL anti-CD3F. Lysed target cells were measured by positive staining for propidium iodide and/or live/dead fixable viability dye. P815 target cells plated without CTLs were used as a negative control (black bar). Primed OTI cells were used as a positive control. Tumors from 10 mice with 2 tumors on opposite flank were pooled to obtain sufficient quantities of CD8⁺ TILs. Data are representative of three independent experiments. (E) Ccl1 and Ccl22 transcript levels in the 4-1BB/LAG-3 subpopulations analyzed directly ex vivo by qRT-PCR. n=4; two independent experiments. *:P<0.05, **:P<0.01, ***:P<0.001, ****:P<0.0001. A Kruskal-Wallis (non-parametric) test was used for (A-C, E) cytokine/chemokine analysis and a two-way ANOVA with Bonferroni post-hoc test was used for (D) cytolytic assay.

FIG. 6A-D. Dysfunctional CD8⁺ TILs express a wide range of co-inhibitory and co-stimulatory receptors. (B) Gene expression profile of cell surface receptors in the 4-1BB/LAG-3 CD8⁺ TIL subsets. Probe sets that revealed a 1.5-fold increase in the 4-1BB⁺LAG-3⁺ population relative to the 4-1BB-LAG-3⁻PD-1⁻ population are displayed. Columns show the log 2-transformed signal intensity. (C) Longitudinal study of selected up-regulated cell surface receptors. Flow plots are representative of the CD8⁺ TIL subsets on day 14. n=5; two to five independent experiments for each time point. (D) Representative flow plot and summary of KLRG-1 and IL-7Rα expression among the 4-1BB/LAG-3 subpopulations on day 14 after tumor inoculation. n=5; two independent experiments. *:P<0.05, **:P<0.01, ***:P<0.001, ****:P<0.0001. A two-way ANOVA with Bonferroni post-hoc test was used for all analyses.

FIG. 7A-G. Anti-4-1BB and anti-LAG-3 acts synergistically to control tumor outgrowth and restore TIL function. (FIG. 7A) Tumor outgrowth measured in mm². Arrows indicate on which days mice received antibody therapy. Statistical significance at indicate time points is in comparison to anti-4-1BB+anti-LAG-3 treatment. n=5; two independent experiments. (FIG. 7B) Composition of H-2K^(b)/SIY⁺ CD8⁺ TILs on day 14. Mice received antibody doses (100 μg each) on days 7, 10, 13 and 16. n=5; two independent experiments. (FIGS. 7C-F) Representative flow plot and summary of NRP1/2B4 (FIGS. 7C and E) and KLRG-1/IL-7Rα (FIGS. 7D and F) expression in H-2K^(b)/SIY⁺ CD8⁺ TILs without FTY720 (FIGS. 7C and D) and with FTY720 (FIGS. 7E and F) on day 14 after tumor inoculation. Mice received antibody treatment as in (FIGS. 7A and B) and FTY720 was administered at a dose of 25 μg/mouse by gavage starting one day before treatment and continuing one dose per day until analysis (day 6 to day 13). n=5; two-independent experiments. (FIG. 7G) IL-2 production after treatment. Sorted cells from treated or untreated day 14 B16. SIY tumor bearing mice were stimulated in vitro for 12 hours and analyzed for Il-2 transcript by qRT-PCR. Protein concentration was determined by the bead-based LEGENDplex immunoassay and normalized to cell number. Two tumors on opposite flanks pooled per mouse. n=2-3; two independent experiments. A two-way ANOVA with Bonferroni post-hoc test was used for all analyses. *:P<0.05, **:P<0.01, ***:P<0.001.

FIG. 8 . Spectratype graphs used in the analysis in FIG. 3B.

FIG. 9 . CD3+ T cells on day 14 after FTY720 administration.

FIG. 10A-B. Statistical analysis of the cross-study comparison of gene expression profiles. (A) Rank-Rank Hypergeometric plots of each pair-wise comparison. (B) Pair-wise correlation of expression values between each data set. Rho (p) is the spearman rank correlation coefficient.

FIG. 11A-E. Nrn1, CRTAM and Sema7a are regulators of anti-tumor immunity. (FIG. 11A) Tumor growth measured in mm2. Nrn1^(−/−) or Sema7a^(−/−) and littermate control mice were engrafted with 2×106 B16. SIY cells subcutaneously. (FIG. 11B) Gene expression analysis of Nrn1 in T cell subsets of the spleen, TdLN and Tumor. (FIG. 11C) Representative flow plot and summary of IFN-g production of WT, Nrn1^(−/−) or (FIG. 11D) CRTAM^(−/−) 2C T cells on day 7. Briefly, on the same day as tumor inoculation, 1×10⁶ Cell Trace Violet-labeled 2C T cells were transferred into mice by tail vein injection. On day 7, whole TdLN suspensions were restimulated with SIY peptide for 12 hours and analyzed for cell trace dilution and IFN-g production. (FIG. 11E) Mice that received 1×10⁶ Nrn1^(−/−) 2C T cells are more likely to exhibit complete tumor control compared to mice that received the same number of WT 2C T cells. Adoptive transfer of T cells was performed the same way as in (FIG. 11C).

FIG. 12 . Exemplary experimental protocol and data.

DEFINITIONS

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.

As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition (e.g., cancer, solid tumor cancer, etc.).

As used herein, an “immune response” refers to the action of a cell of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a subject of invading pathogens, cells or tissues infected with pathogens, or cancerous or other abnormal cells.

As used herein, the term “immunoregulator” refers to a substance, an agent, a signaling pathway or a component thereof that regulates an immune response. “Regulating,” “modifying” or “modulating” an immune response refers to any alteration in a cell of the immune system or in the activity of such cell. Such regulation includes stimulation or suppression of the immune system which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes which can occur within the immune system. Both inhibitory and stimulatory immunoregulators have been identified, some of which may have enhanced function in the cancer microenvironment.

As used herein, the term “immunotherapy” refers to the treatment or prevention of a disease or condition by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.

As used herein, “potentiating an endogenous immune response” means increasing the effectiveness or potency of an existing immune response in a subject. This increase in effectiveness and potency may be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

As used herein, the term “antibody” refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab′, and F(ab′)₂), unless otherwise specified (e.g., “whole antibody,” “antibody fragment”). An antibody may be a polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc.

A native antibody typically has a tetrameric structure. A tetramer typically comprises two identical pairs of polypeptide chains, each pair having one light chain (in certain embodiments, about 25 kDa) and one heavy chain (in certain embodiments, about 50-70 kDa). In a native antibody, a heavy chain comprises a variable region, V_(H), and three constant regions, C_(H1), C_(H2), and C_(H3). The V_(H) domain is at the amino-terminus of the heavy chain, and the C_(H3) domain is at the carboxy-terminus. In a native antibody, a light chain comprises a variable region, V_(L), and a constant region, C_(L). The variable region of the light chain is at the amino-terminus of the light chain. In a native antibody, the variable regions of each light/heavy chain pair typically form the antigen binding site. The constant regions are typically responsible for effector function.

In a native antibody, the variable regions typically exhibit the same general structure in which relatively conserved framework regions (FRs) are joined by three hypervariable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDRs on the heavy chain are referred to as H1, H2, and H3, while the CDRs on the light chain are referred to as L1, L2, and L3. Typically, CDR3 is the greatest source of molecular diversity within the antigen-binding site. H3, for example, in certain instances, can be as short as two amino acid residues or greater than 26. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat et al. (1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Publication No. 91-3242, vols. 1-3, Bethesda, Md.); Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196:901-917; or Chothia, C. et al. Nature 342:878-883 (1989). In the present application, the term “CDR” refers to a CDR from either the light or heavy chain, unless otherwise specified.

As used herein, the term “heavy chain” refers to a polypeptide comprising sufficient heavy chain variable region sequence to confer antigen specificity either alone or in combination with a light chain.

As used herein, the term “light chain” refers to a polypeptide comprising sufficient light chain variable region sequence to confer antigen specificity either alone or in combination with a heavy chain.

As used herein, when an antibody or other entity “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (K_(a)) of at least 10⁷ M⁻¹ (e.g., >10⁷ M⁻¹, >10⁸ M⁻¹, >10⁹ M⁻¹, >10¹⁰ M⁻¹, >10¹¹ M⁻¹, >10¹² M⁻¹, >10¹³ M⁻¹, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

As used herein, the term “anti-4-1BB antibody” or “4-1BB antibody” refers to an antibody which specifically recognizes an antigen and/or epitope presented by 4-1BB. Similarly, the terms “anti-LAG-3 antibody” and “LAG-3 antibody” refer to an antibody which specifically recognizes an antigen and/or epitope presented by LAG-3, the terms “anti-Nrn1 antibody” and “Nrn1 antibody” refer to an antibody which specifically recognizes an antigen and/or epitope presented by Nrn1, the terms “anti-CRTAM antibody” and “CRTAM antibody” refer to an antibody which specifically recognizes an antigen and/or epitope presented by CRTAM, and the terms “anti-Sema7a antibody” and “Sema7a antibody” refer to an antibody which specifically recognizes an antigen and/or epitope presented by Sema7a. Antibodies that recognize epitopes on other molecular entities may be referred to according to a similar scheme (e.g., anti-CTLA-4, anti-PD-L1, etc.).

As used herein, the term “monoclonal antibody” refers to an antibody which is a member of a substantially homogeneous population of antibodies that specifically bind to the same epitope. In certain embodiments, a monoclonal antibody is secreted by a hybridoma. In certain such embodiments, a hybridoma is produced according to certain methods known to those skilled in the art. See, e.g., Kohler and Milstein (1975) Nature 256: 495-499; herein incorporated by reference in its entirety. In certain embodiments, a monoclonal antibody is produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In certain embodiments, a monoclonal antibody refers to an antibody fragment isolated from a phage display library. See, e.g., Clackson et al. (1991) Nature 352: 624-628; and Marks et al. (1991) J. Mol. Biol. 222: 581-597; herein incorporated by reference in their entireties. The modifying word “monoclonal” indicates properties of antibodies obtained from a substantially-homogeneous population of antibodies, and does not limit a method of producing antibodies to a specific method. For various other monoclonal antibody production techniques, see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); herein incorporated by reference in its entirety.

As used herein, the term “antibody fragment” refers to a portion of a full-length antibody, including at least a portion antigen binding region or a variable region. Antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; herein incorporated by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain digestion and pepsin digestion of antibody) produced by recombinant DNA techniques, or chemical polypeptide synthesis.

For example, a “Fab” fragment comprises one light chain and the C_(H1) and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab′” fragment comprises one light chain and one heavy chain that comprises additional constant region, extending between the C_(H1) and CH₂ domains. An interchain disulfide bond can be formed between two heavy chains of a Fab′ fragment to form a “F(ab′)₂” molecule.

An “Fv” fragment comprises the variable regions from both the heavy and light chains, but lacks the constant regions. A single-chain Fv (scFv) fragment comprises heavy and light chain variable regions connected by a flexible linker to form a single polypeptide chain with an antigen-binding region. Exemplary single chain antibodies are discussed in detail in WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203; herein incorporated by reference in their entireties. In certain instances, a single variable region (e.g., a heavy chain variable region or a light chain variable region) may have the ability to recognize and bind antigen.

Other antibody fragments will be understood by skilled artisans.

As used herein, the term “chimeric antibody” refers to an antibody made up of components from at least two different sources. In certain embodiments, a chimeric antibody comprises a portion of an antibody derived from a first species fused to another molecule, e.g., a portion of an antibody derived from a second species. In certain such embodiments, a chimeric antibody comprises a portion of an antibody derived from a non-human animal fused to a portion of an antibody derived from a human. In certain such embodiments, a chimeric antibody comprises all or a portion of a variable region of an antibody derived from a non-human animal fused to a constant region of an antibody derived from a human.

A “humanized” antibody refers to a non-human antibody that has been modified so that it more closely matches (in amino acid sequence) a human antibody. A humanized antibody is thus a type of chimeric antibody. In certain embodiments, amino acid residues outside of the antigen binding residues of the variable region of the non-human antibody are modified. In certain embodiments, a humanized antibody is constructed by replacing all or a portion of a complementarity determining region (CDR) of a human antibody with all or a portion of a CDR from another antibody, such as a non-human antibody, having the desired antigen binding specificity. In certain embodiments, a humanized antibody comprises variable regions in which all or substantially all of the CDRs correspond to CDRs of a non-human antibody and all or substantially all of the framework regions (FRs) correspond to FRs of a human antibody. In certain such embodiments, a humanized antibody further comprises a constant region (Fc) of a human antibody.

The term “human antibody” refers to a monoclonal antibody that contains human antibody sequences and does not contain antibody sequences from a non-human animal. In certain embodiments, a human antibody may contain synthetic sequences not found in native antibodies. The term is not limited by the manner in which the antibodies are made. For example, in various embodiments, a human antibody may be made in a transgenic mouse, by phage display, by human B-lymphocytes, or by recombinant methods.

As used herein, the term “natural antibody” refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multicellular organism. For example, the antibodies produced by the antibody-producing cells isolated from a first animal immunized with an antigen are natural antibodies. Natural antibodies contain naturally-paired heavy and light chains. The term “natural human antibody” refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a human subject.

Native human light chains are typically classified as kappa and lambda light chains. Native human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA has subclasses including, but not limited to, IgA1 and IgA2.

Within native human light and heavy chains, the variable and constant regions are typically joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology (1989) Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.); herein incorporated by reference in its entirety.

The term “neutralizing antibody” or “antibody that neutralizes” refers to an antibody that reduces at least one activity of a polypeptide comprising the epitope to which the antibody specifically binds. In certain embodiments, a neutralizing antibody reduces an activity in vitro and/or In vivo. In some embodiments, by neutralizing the polypeptide comprising the epitope, the neutralizing antibody inhibits the capacity of the cell displaying the epitope.

As used herein, the term “glycoengineered”, as used herein, includes any manipulation of the glycosylation pattern of a naturally occurring or recombinant protein, polypeptide or a fragment thereof.

The term “antigen-binding site” refers to a portion of an antibody capable of specifically binding an antigen. In certain embodiments, an antigen-binding site is provided by one or more antibody variable regions.

The term “epitope” refers to any polypeptide determinant capable of specifically binding to an immunoglobulin or a T-cell or B-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In certain embodiments, an epitope may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three dimensional structural characteristics (e.g., a “conformational” epitope) and/or specific charge characteristics.

An epitope is defined as “the same” as another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides having different primary amino acid sequences may comprise epitopes that are the same. In certain embodiments, epitopes that are the same may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope if they compete for specific binding to that epitope.

A “conservative” amino acid substitution refers to the substitution of an amino acid in a polypeptide with another amino acid having similar properties, such as size or charge. In certain embodiments, a polypeptide comprising a conservative amino acid substitution maintains at least one activity of the unsubstituted polypeptide. A conservative amino acid substitution may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Naturally occurring residues may be divided into classes based on common side chain properties, for example: hydrophobic: norleucine, Met, Ala, Val, Leu, and Ile; neutral hydrophilic: Cys, Ser, Thr, Asn, and Gln; acidic: Asp and Glu; basic: His, Lys, and Arg; residues that influence chain orientation: Gly and Pro; and aromatic: Trp, Tyr, and Phe. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class; whereas conservative substitutions may involve the exchange of a member of one of these classes for another member of that same class.

As used herein, the term “sequence identity” refers to the degree to which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families (see above). The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.

The term “effective dose” or “effective amount” refers to an amount of an agent, e.g., an antibody, that results in the reduction of symptoms in a patient or results in a desired biological outcome. In certain embodiments, an effective dose or effective amount is sufficient to treat or reduce symptoms of a disease or condition.

As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

The term “treatment” encompasses both therapeutic and prophylactic/preventative measures unless otherwise indicated. Those in need of treatment include, but are not limited to, individuals already having a particular condition as well as individuals who are at risk of acquiring a particular condition or disorder (e.g., those having a genetic or epigenetic predisposition; based on age, gender, lifestyle, etc.). The term “treating” refers to administering an agent to a subject for therapeutic and/or prophylactic/preventative purposes.

A “therapeutic agent” refers to an agent that may be administered In vivo to bring about a therapeutic and/or prophylactic/preventative effect.

A “therapeutic antibody” refers to an antibody that may be administered In vivo to bring about a therapeutic and/or prophylactic/preventative effect.

As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

As used herein, the term pharmaceutical composition” refers to the combination of an active agent (e.g., binding agent) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintigrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference in its entirety.

As used herein, a “diagnostic” or “diagnostic test” includes the detection, identification, or characterization of a disease state or condition of a subject. For example, a disease or condition may be characterized to determine the likelihood that a subject with a disease or condition will respond to a particular therapy, determine the prognosis of a subject with a disease or condition (or its likely progression or regression), determine the effect of a treatment on a subject with a disease or condition, or determine a future treatment course of action.

DETAILED DESCRIPTION

Provided herein are compositions and methods for detecting and/or targeting dysfunctional tumor antigen-specific CD8⁺ T cells in the tumor microenvironment for diagnostic, therapeutic and/or research applications. In particular, dysfunctional tumor antigen-specific CD8⁺ T cells are detected and/or targeted via their expression of cell surface receptors described herein, such as 4-1BB, LAG-3, or additional markers that correlate with 4-1BB and LAG-3 expression, such as markers differentially expressed on the surface of the T cells (e.g., PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM and Sema7a).

Experiments conducted during development of embodiments herein identified markers/receptors that correlate and/or are responsible for tumor antigen-specific CD8⁺ T cell dysfunction. In some embodiments, the markers/receptors are overexpressed in dysfunctional tumor antigen-specific CD8⁺ T cells. In such embodiments, detecting the level (e.g., above a threshold) of such markers provides a diagnostic for detecting tumor antigen-specific CD8⁺ T cell dysfunction. Further, in such embodiments, targeting (e.g., inhibiting (e.g., expression and/or activity of)) such markers/receptors provides a therapeutic. In other embodiments, the markers/receptors are underexpressed in dysfunctional tumor antigen-specific CD8⁺ T cells. In such embodiments, detecting the level (e.g., below a threshold) of such markers provides a diagnostic for detecting tumor antigen-specific CD8⁺ T cell dysfunction. Further, in such embodiments, targeting (e.g., enhancing (e.g., expression and/or activity of)) such markers/receptors provides a therapeutic.

Transcription factor Egr2 is a critical regulator of the anergic state in CD4⁺ T cell clones manipulated in vitro (Zheng et al., 2013; 2012; incorporated by reference in their entireties). Egr2 has also been shown to be involved in negative regulation of T cell activation in several in vivo model systems (Sumitomo et al., 2013; incorporated by reference in its entirety). Egr2 contributes to upregulation of DGKa and -z which act to blunt TCR-mediated Ras pathway activation (Zha et al., 2006; incorporated by reference in its entirety). By comparing gene expression profiling of anergized cells along with Egr2 ChIP-Seq analysis multiple additional Egr2-driven gene targets were identified (Zheng et al., 2013; incorporated by reference in its entirety). These gene targets include 4-1BB (Tnfrsf9 or CD137), Lag3, Nrn1, Sema7a, Crtam, and Rank1, which encode cell surface proteins. 4-1BB is a co-stimulatory molecule transiently expressed after TCR engagement. Lag3 (lymphocyte-activation gene 3 or CD223) is a CD4 homologue and functions as an inhibitory receptor. Expression of 4-1BB and Lag3 is regulated following TCR engagement and continues throughout differentiation. In humans, 4-1BB and LAG-3 are expressed on CD8+ TILs from human melanoma tumors (Gros et al., 2014; Baitsch et al., 2012; incorporated by reference in their entireties). In both mice and humans, either molecule alone are expressed on populations of activated T cells. However, co-expression is more limited and is rarely observed in circulating T cells. The function of CD8+ TILs co-expressing these markers is unknown.

Experiments were conducted during development of embodiments herein to investigate the detailed characteristics of CD8+ TILs expressing 4-1BB and LAG-3 using mouse tumor models. It was found that the co-expression of 4-1BB and LAG-3 was sufficient to identify tumor antigen-specific dysfunctional CD8+ TILs enriched in the expression of Egr2 target genes. These CD8+ TILs failed to make IL-2 following in vitro stimulation, yet still produced IFN-g and Treg-recruiting chemokines and lysed target cells ex vivo, indicating they are not completely functionally inert. Combinatorial treatment with anti-LAG-3/anti-4-1BB restored the function of this population and promoted in situ acquisition of KLRG-1hi effector cells. Additional gene expression profiling provided a complete phenotyping of this T cell subset, which revealed expression of a broad panel of both inhibitory receptors and co-stimulatory receptors (e.g., receptors of Table 2 (e.g. Nrn1, Sema7a, CRTAM, etc.)). Inhibitory receptors and co-stimulatory receptors identified in this profiling that are displayed on the surface of T cells include PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A. These approaches have thus enabled the characterization of the population of tumor antigen-specific CD8+ T cells that arise specifically within the tumor microenvironment having altered functional properties. In some embodiments, this population is a target for immunotherapeutic approaches to restore desired functionality and promote tumor regression. In some embodiments, the receptors/markers identified herein (e.g., 4-1BB, LAG-3, receptors/markers of Table 2 (e.g., surface markers/receptors (e.g. Nrn1, Sema7a, CRTAM, etc.), etc.) etc.) are targeted (e.g., via immunotherapeutic approaches) to restore desired immunoresponsiveness, to promote tumor regression, and/or for the treatment of cancer.

Experiments conducted during development of embodiments herein applied knowledge of Egr2 targets to evaluate applicability of these markers toward understanding dysfunctional T cells within tumors in vivo. The data indeed confirm that co-expression of LAG-3 and 4-1BB is sufficient to identify the majority of tumor antigen-specific CD8⁺ T cells within the tumor microenvironment. Co-expression of these markers was not observed within peripheral lymphoid organs in tumor-bearing mice, indicating that a property unique to the tumor context drives 4-1BB and LAG-3 expression. In addition, acquisition of LAG-3 and 4-1BB expression was not observed within tumors that were undergoing successful rejection, indicating that the acquisition of this phenotype occurs under conditions of incomplete antigen clearance.

In some embodiments, cancer treatment methods described herein comprise administration (or co-administration with one or more additional therapies/therapeutics) of one or more anti-4-1BB and/or anti-LAG-3 agents (e.g., antibodies, antibody fragments, antibody mimetic molecules (e.g., DARPins, affibodies, aptamers, nanobodies, etc.), etc.). In some embodiments, an anti-4-1BB and/or anti-LAG-3 agents is administered to render cancer cells, tumor(s), and/or the tumor microenvironment accessible or susceptible to treatment with additional therapies/therapeutics (e.g., immunotherapeutics). Anti-4-1BB and/or anti-LAG-3 agents that find use in embodiments described herein are not limited by their mechanism of action. Agents may be small molecules, peptide, polypeptides, proteins, nucleic acids (e.g., antisense, RNAi, etc.), antibodies, antibody fragments, etc.

In some embodiments, cancer treatment methods described herein comprise enhancing the activity or expression of a marker/receptor identified herein that negatively correlates with tumor antigen-specific CD8⁺ T cell dysfunction.

Experiments conducted during development of embodiments herein identified receptors/markers that are differentially expressed in dysfunctional CD8+ TILs (See Table 2). Testing of targets of interest identified in that screen demonstrate that at least neuritin 1 (Nrn1), cytotoxic and regulatory t-cell molecule (CRTAM), and Semaphorin 7A (Sema7a) are regulators of anti-tumor immunity, with Nrn1 and CRTAM blockade correlating with increased tumor area, and Sema7a blockade correlating with decreased tumor area.

In some embodiments, cancer treatment methods described herein comprise administration (or co-administration with one or more additional therapies/therapeutics) of agents (e.g., antibodies, antibody fragments, antibody mimetic molecules (e.g., DARPins, affibodies, aptamers, nanobodies, etc.), etc.) that target one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.). In some embodiments, an agent is administered to render cancer cells, tumor(s), and/or the tumor microenvironment accessible or susceptible to treatment with additional therapies/therapeutics (e.g., immunotherapeutics). Agents targeting one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.) that find use in embodiments described herein are not limited by their mechanism of action. Agents may be small molecules, peptide, polypeptides, proteins, nucleic acids (e.g., antisense, RNAi, etc.), antibodies, antibody fragments, etc. In some embodiments, an antagonist of Nrn1 is administered. In some embodiments, an antagonist of CRTAM is administered. In some embodiments, an agonist of Sema7a is administered.

In some embodiments, antibodies, antibody fragments, antibody mimetic molecules (e.g., DARPins, affibodies, aptamers, nanobodies, etc.) targeting 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.), or fragments thereof, are provided. Such agents may be naked, deriving their effect by target binding (e.g., neutralizing the target), or may be conjugated to a functional moiety (e.g., drug, toxin, effector moiety, etc.).

In some embodiments, a subject is treated with (i) one or more agents (e.g., antibodies, antibody fragments, antibody mimetic molecules (e.g., DARPins, affibodies, aptamers, nanobodies, etc.), etc.) that target 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.), as well as (ii) one or more additional cancer therapies. Such therapies include chemotherapy, immunotherapy, radiation, surgery, etc. In some embodiments, agents targeting the receptors/markers described herein are co-administered with one or more additional agents for the treatment of cancer.

In some embodiments, exemplary anticancer agents suitable for use in compositions and methods described herein include, but are not limited to: 1) alkaloids, including microtubule inhibitors (e.g., vincristine, vinblastine, and vindesine, etc.), microtubule stabilizers (e.g., paclitaxel (Taxol), and docetaxel, etc.), and chromatin function inhibitors, including topoisomerase inhibitors, such as epipodophyllotoxins (e.g., etoposide (VP-16), and teniposide (VM-26), etc.), and agents that target topoisomerase I (e.g., camptothecin and isirinotecan (CPT-11), etc.); 2) covalent DNA-binding agents (alkylating agents), including nitrogen mustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide, ifosphamide, and busulfan (MYLERAN), etc.), nitrosoureas (e.g., carmustine, lomustine, and semustine, etc.), and other alkylating agents (e.g., dacarbazine, hydroxymethylmelamine, thiotepa, and mitomycin, etc.); 3) noncovalent DNA-binding agents (antitumor antibiotics), including nucleic acid inhibitors (e.g., dactinomycin (actinomycin D), etc.), anthracyclines (e.g., daunorubicin (daunomycin, and cerubidine), doxorubicin (adriamycin), and idarubicin (idamycin), etc.), anthracenediones (e.g., anthracycline analogues, such as mitoxantrone, etc.), bleomycins (BLENOXANE), etc., and plicamycin (mithramycin), etc.; 4) antimetabolites, including antifolates (e.g., methotrexate, FOLEX, and MEXATE, etc.), purine antimetabolites (e.g., 6-mercaptopurine (6-MP, PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir, chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and 2′-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g., fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL), 5-fluorodeoxyuridine (FdUrd) (floxuridine)) etc.), and cytosine arabinosides (e.g., CYTOSAR (ara-C) and fludarabine, etc.); 5) enzymes, including L-asparaginase, and hydroxyurea, etc.; 6) hormones, including glucocorticoids, antiestrogens (e.g., tamoxifen, etc.), nonsteroidal antiandrogens (e.g., flutamide, etc.), and aromatase inhibitors (e.g., anastrozole (ARIMIDEX), etc.); 7) platinum compounds (e.g., cisplatin and carboplatin, etc.); 8) monoclonal antibodies (e.g., conjugated with anticancer drugs, toxins, and/or radionuclides, etc.; neutralizing antibodies; etc.); 9) biological response modifiers (e.g., interferons (e.g., IFN-.alpha., etc.) and interleukins (e.g., IL-2, etc.), etc.); 10) adoptive immunotherapy; 11) hematopoietic growth factors; 12) agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14) antisense therapy techniques; 15) tumor vaccines; 16) therapies directed against tumor metastases (e.g., batimastat, etc.); 17) angiogenesis inhibitors; 18) proteosome inhibitors (e.g., VELCADE); 19) inhibitors of acetylation and/or methylation (e.g., HDAC inhibitors); 20) modulators of NF kappa B; 21) inhibitors of cell cycle regulation (e.g., CDK inhibitors); and 22) modulators of p53 protein function.

In some embodiments, agents targeting 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. Nrn1, Sema7a, CRTAM, etc.) are administered to overcome immune invasion of the cancer cells, tumor, tumor microenvironment, etc. In some embodiments, one or more additional cancer immunotherapies are employed (e.g., concurrently or serially) to make use of the immune-responsiveness of the treated cells/tumor. Suitable immunotherapies may include, but are not limited to: cell-based therapies (e.g., dendritic cell or T cell therapy, etc.), monoclonal antibody (mAb) therapy (e.g., naked mAbs, conjugated mAbs), cytokine therapy (e.g., interferons, interleukins, etc.), adjuvant treatment (e.g., polysaccharide-K), etc.

In some embodiments, agents targeting 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.) are co-administered with agents (e.g., small molecules, peptides, antibodies, antibody fragments, etc.) that target one or more cancer cell or tumor) markers or components. In some embodiments, such co-administration renders the cancer cells, tumor, and/or tumor microenvironment susceptible and/or accessible to the treatment with the additional agent.

In some embodiments, agents for use in the methods and compositions described herein target and/or binds a cancer or tumor cell marker or component, selected from the group including but not limited to, epidermal growth factor receptor (EGFR, EGFR1, ErbB-1, HER1). ErbB-2 (HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF-binding proteins (IGFBPs), IGFR ligand family (IGF-1R); platelet derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family, VEGF family; HGF receptor family: TRK receptor family; ephrin (EPH) receptor family: AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family; discoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; Transforming growth factor alpha (TGF-α), TGF-α receptor; Transforming growth factor-beta (TGF-β), TGF-β receptor; Interleukin β receptor alpha2 chain (IL13Ralpha2), Interleukin-6 (IL-6), 1L-6 receptor, interleukin-4, IL-4 receptor, Cytokine receptors, Class I (hematopoietin family) and Class II (interferon/1L-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNTRSF), death receptor family, TRAIL-receptor; cancer-testis (CT) antigens, lineage-specific antigens, differentiation antigens, alpha-actinin-4, ARTC1, breakpoint cluster region-Abelson (Bcr-abl) fusion products, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), beta-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, Elongation factor 2 (ELF2), Ets variant gene 6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low density lipid receptor/GDP-L fucose: beta-Dgalactose 2-alpha-Lfucosyltraosferase (LDLR/FUT) fusion protein, HLA-A2, MLA-A11, heat shock protein 70-2 mutated (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous mutated 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, Myosin class 1, NFYC, OGT, OS-9, pm1-RARalpha fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT12, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, Triosephosphate Isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V (aberrant N-acetyl glucosaminyl transferase V, MGAT5), HERV-K MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized antigen on melanoma (CAMEL), MAGE-A1 (MAGE-1). MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10. MAGE-All, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5. MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1), MART-1/Melan-A (MLANA), gp100, gp100/Pme117 (SlLV), tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2, SAGE, Sp17. SSX-1, 2, 3, 4, TRP2-1NT2, carcino-embryonic antigen (CEA), Kallikrein 4, mammaglobin-A, OA1, prostate specific antigen (PSA), prostate specific membrane antigen, TRP-1/, 75. TRP-2 adipophilin, interferon inducible protein absent in melanora 2 (AIM-2). BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250intestinal carboxyl esterase (iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUCI, p53 (TP53), PBF, PRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, survivin (BIRCS), human telomerase reverse transcriptase (hTERT), telomerase, Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE, immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors (ER), androgen receptors (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), beta-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96. GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinoembryogenic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), etc.

Examples of antibodies which can be incorporated into compositions and methods disclosed herein include, but are not limited, to antibodies such as trastuzumab (anti-HER2/neu antibody); Pertuzumab (anti-HER2 mAb); cetuximab (chimeric monoclonal antibody to epidermal growth factor receptor EGFR); panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFR antibody); Zalutumumab (anti-EGFR mAb); Necitumumab (anti-EGFR mAb); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptor bispecific antibody); Rituximab (chimeric murine/human anti-CD20 mAb); Obinutuzumab (anti-CD20 mAb); Ofatumumab (anti-CD20 mAb); Tositumumab-1131 (anti-CD20 mAb); Ibritumomab tiuxetan (anti-CD20 mAb); Bevacizumab (anti-VEGF mAb); Ramucirumab (anti-VEGFR2 mAb); Ranibizumab (anti-VEGF mAb); Aflibercept (extracellular domains of VEGFR1 and VEGFR2 fused to IgG1 Fc); AMG386 (angiopoietin-1 and -2 binding peptide fused to IgG1 Fc); Dalotuzumab (anti-IGF-1R mAb); Gemtuzumab ozogamicin (anti-CD33 mAb); Alemtuzumab (anti-Campath-1/CD52 mAb); Brentuximab vedotin (anti-CD30 mAb): Catumaxomab (bispecific mAb that targets epithelial cell adhesion molecule and CD3); Naptumomab (anti-5T4 mAb); Girentuximab (anti-Carbonic anhydrase ix); or Farletuzumab (anti-folate receptor). Other examples include antibodies such as Panorex™ (17-1A) (murine monoclonal antibody); Panorex (@(17-1A)) (chimeric murine monoclonal antibody); BEC2 (ami-idiotypic mAb, mimics the GD epitope) (with BCG); Oncolym (Lym-1 monoclonal antibody); SMART M195 Ab, humanized 13′ 1 LYM-1 (Oncolym). Ovarex (B43.13, anti-idiotypic mouse mAb); 3622W94 mAb that binds to EGP40 (17-1A) pancarcinoma antigen on adenocarcinomas; Zenapax (SMART Anti-Tac (IL-2 receptor); SMART M195 Ab, humanized Ab, humanized); NovoMAb-G2 (pancarcinoma specific Ab); TNT (chimeric mAb to histone antigens); TNT (chimeric mAb to histone antigens); Gliomab-H (Monoclonals—Humanized Abs); GNI-250 Mab; EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanized IL. L.2 antibody); and MDX-260 bispecific, targets GD-2, ANA Ab, SMART IDIO Ab, SMART ABL 364 Ab, or ImmuRAIT-CEA.

In some embodiments, an agent that finds use in embodiments herein specifically binds a component of a regulatory T cell, myeloid suppressor cell, or dendritic cell. In another aspect, the targeting moiety specifically binds one of the following molecules: CD4; CD25 (IL-2a receptor; IL-2αR); cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152); Interleukin-10 (IL-10); Transforming growth factor-beta receptor (TGF-βR); Transforming growth factor-beta (TGF-β); Programmed Death-1 (PD-1); Programmed death-1 ligand (PD-L1 or PD-L2); Receptor activator of nuclear factor-κB (RANK); Receptor activator of nuclear factor-κB (RANK) ligand (RANKL); LAG-3; glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR; TNFRSF18); or Interleukin-4 receptor (IL-4R). In some embodiments, the agent is an agonist that increases the function of the targeted molecule. In other embodiments, the agent is an antagonist that inhibits the function of the targeted molecule.

In some embodiments, an agent that finds use in embodiments herein binds a specific cytokine, cytokine receptor, co-stimulatory molecule, co-inhibitory molecule, or immunomodulatory receptor that modulates the immune system. In another aspect, the targeting moiety specifically binds one of the following molecules: tumor necrosis factor (TNF) superfamily; tumor necrosis factor-α (TNF-α); tumor necrosis factor receptor (TNFR) superfamily; Interleukin-12 (IL-12); IL-12 receptor; 4-1BB (CD137); 4-1BB ligand (4-1BBL; CD137L); OX40 (CD134; TNR4); OX40 ligand (OX40L; CD40; CD40 ligand (CD40L); CTLA-4; Programmed death-1 (PD-1); PD-1 ligand I (PD-L1: B7-H1); or PD-1 ligand 2 (PD-L2; B7-DC); B7 family; B7-1 (CD80); B7-2 (CD86); B7-H3; B7-H4; GITR/AITR: GITRL/AITRL; BTLA; CD70; CD27; LIGHT; HVEM: Toll-like receptor (TLR) (TLR 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, the agent is an agonist that increases the function of the targeted molecule. In other embodiments, the agent is an antagonist that inhibits the function of the targeted molecule.

In some embodiments, agents (e.g., immunotherapeutics) targeting 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.) are co-administered (e.g., serially or sequentially) with one or more adjuvants. Suitable adjuvants include, but are not limited to, one or more of: oil emulsions (e.g., Freund's adjuvant); saponin formulations; virosomes and viral-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., mineral salts, such as aluminium salts and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and/or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene; muramyl peptides; imidazoquinolone compounds; and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-.gamma.), macrophage colony stimulating factor, and tumor necrosis factor. In addition to variant B7-DC polypeptides, other co-stimulatory molecules, including other polypeptides of the B7 family, may be administered. Proteinaceous adjuvants may be provided as the full-length polypeptide or an active fragment thereof, or in the form of DNA, such as plasmid DNA.

Pharmaceutical and immunotherapeutic compositions described herein may be delivered by any suitable route of administration (e.g., oral delivery, parenteral delivery, mucous membrane delivery, pulmonary delivery, intravenous delivery, etc.). Appropriate formulations for such delivery routes are understood in the field.

Non-limiting examples of cancers that may be treated with the compositions and methods described herein include, but are not limited to: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), pancreatic cancer (e.g., adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g. non-small cell lung cancer), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies. In some embodiments, the cancer is a solid tumor cancer.

Some embodiments described herein are particularly useful for the treatment of tumors that do not otherwise respond to immunotherapeutic approaches. In some embodiments, provided herein is the treatment of cancers that are non-responsive (or have a reduced response) to T cells or antigen presenting cells (e.g., dendritic cells (e.g., CD103⁺DCs, etc.), etc.). In some embodiments, provided herein is the treatment of cancers that are non-responsive to treatments, despite T cell infiltration. In some embodiments, compositions and methods described herein find use in the treatment of cancers in which T cells are not appropriately primed against tumor-associated antigens. In some embodiments, compositions and methods described herein find use in the treatment of cancers comprising tumors or cells that are defective in recruitment of dendritic cells (e.g., CD103⁺ DCs, etc.). In some embodiments, compositions and methods described herein find use in the treatment of cancers comprising tumors or cells that are defective in production of the chemokine CCL4.

In some embodiments, the therapeutic compositions and methods herein find use with those described in, for example WO 2016/141312; incorporated by reference in its entirety.

In some embodiments, methods are provided for testing sample (e.g., cell, tissue, population of cells, tumor, blood, urine, saliva, etc.) from a subject for one or more biomarkers (e.g., biomarkers of dysfunctional tumor antigen-specific CD8⁺ T cells). Such biomarkers may comprise nucleic acids, small molecules, proteins, peptides, etc., and may be detected using any suitable assay of technique. In some embodiments, provided herein are DNA-, RNA-, small molecule, and/or protein-based diagnostic methods that either directly or indirectly detect the biomarkers of the evasion of immune response or immunotherapy by cancer cells or tumors. The present invention also provides compositions, reagents, and kits for such diagnostic purposes.

In some embodiments, biomarkers are detected at the nucleic acid (e.g., RNA) level. For example, the presence or amount of biomarker nucleic acid (e.g., mRNA) in a sample is determined (e.g., to determine the presence or level of biomarker expression). Biomarker nucleic acid (e.g., RNA, amplified cDNA, etc.) may be detected/quantified using a variety of nucleic acid techniques known to those of ordinary skill in the art, including but not limited to nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification (e.g., by PCR, RT-PCR, qPCR, etc.), micorarray, Southern and Northern blotting, sequencing, etc. Non-amplified or amplified nucleic acids can be detected by any conventional means. For example, in some embodiments, nucleic acids are detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids. Nucleic acid detection reagents may be labeled (e.g., fluorescently) or unlabeled, and may by free in solution or immobilized (e.g., on a bead, well, surface, chip, etc.).

In some embodiments, biomarkers are detected at the protein level. For example, the presence or amount of biomarker protein in a sample is determined (e.g., to determine the presence or level of biomarker expression or localization). In some embodiments, reagents are provided for the detection and/or quantification of biomarker proteins. Suitable reagents include primary antibodies (e.g., that bind to the biomarkers), secondary antibodies (e.g., that bind primary antibodies), antibody fragments, aptamers, etc. Protein detection reagents may be labeled (e.g., fluorescently) or unlabeled, and may by free in solution or immobilized (e.g., on a bead, well, surface, chip, etc.).

In some embodiments, biomarker capture reagents are provided to localize, concentrate, aggregate, etc. a biomarker. For example, in some embodiments a biomarker capture reagent that interacts with the biomarker is linked to a solid support (e.g., a bead, surface, resin, column, and the like) that allows manipulation by the user on a macroscopic scale. Often, the solid support allows the use of a mechanical means to isolate and purify the biomarker from a heterogeneous solution. For example, when linked to a bead, separation is achieved by removing the bead from the heterogeneous solution, e.g., by physical movement. In embodiments in which the bead is magnetic or paramagnetic, a magnetic field is used to achieve physical separation of the capture reagent (and thus the target) from the heterogeneous solution. Magnetic beads used to isolate targets are described in the art, e.g., as described in European Patent Application No. 87309308, incorporated herein in its entirety for all purposes.

Compositions for use in the diagnostic methods or testing steps described herein include, but are not limited to, probes, amplification oligonucleotides, and antibodies. Any of the detection and/or diagnostic reagents used in embodiments described herein may be provided alone or in combination with other compositions in the form of a kit. Kits may include any and all components necessary or sufficient for assays including, but not limited to, the detection reagents, buffers, control reagents (e.g., tissue samples, positive and negative control sample, etc.), solid supports, labels, written and/or pictorial instructions and product information, inhibitors, labeling and/or detection reagents, package environmental controls (e.g., ice, desiccants, etc.), and the like. In some embodiments, the kits provide a sub-set of the required components, wherein it is expected that the user will supply the remaining components. In some embodiments, the kits comprise two or more separate containers wherein each container houses a subset of the components to be delivered.

In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of expression a biomarker) into data of predictive value for a clinician. In some embodiments, computer analysis combines various data into a single score or value that is predictive and/or diagnostic. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject. Contemplated herein are any methods capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information providers, medical personal, and subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy, cell, or blood sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, third-party testing service, genomic profiling business, etc. to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves and directly send it to a profiling center. In some embodiments, a report is generated (e.g., by a clinician, by a testing center, by a computer or other automated analysis system, etc.). A report may contain test results, diagnoses, and/or treatment recommendations.

EXPERIMENTAL Materials and Methods Mice and Tumor Inoculation

Female C57BL/6 mice ranging from 6 to 8 weeks were purchased from Taconic Farms. CD45.1 and Rag2^(−/−) mice on the C57BL/6 background were obtained from Taconic Farms and bred at the University of Chicago. 2C/Rag2^(−/−) and P14/Rag2^(−/−) mice have been previously described (Brown et al., 2006; incorporated by reference in its entirety). pLCK-CreERT2×ROSA-YFP mice were generated and have been described (Evaristo et al., 2016; incorporated by reference in its entirety). B16. SIY.dsRed (Kline et al., 2012; incorporated by reference in its entirety), C1498. SIY. GFP (Zhang et al., 2009; incorporated by reference in its entirety), and MC57. SIY. GFP (Spiotto et al., 2002; incorporated by reference in its entirety) tumor cells were engineered to express either dsRed or GFP in frame with the H2-K^(b)-restricted model antigen SIYRYYGL. The 1969. SIY. GFP cell line was engineered by retroviral transduction of the 1969 cell line (Diamond et al., 2011; incorporated by reference in its entirety) using the pLEGFP plasmid expressing cDNA for SIYRYYGL (Spiotto et al., 2002; incorporated by reference in its entirety). For experiments, mice 6 to 9 weeks of age and received 2×10⁶ tumor cells subcutaneously on either the left flank or both the left and right flank. All mice were maintained according to the National Institute of Health Animal Care guidelines and studied under IACUC-approved protocols.

To generate the targeting construct for the Egr2^(EGFP) knock-in reporter mice, a 12.6 kb mouse genomic DNA fragment including the egr2 gene was excised with SacII and cloned into a pEasy-Flox vector adjacent to the thymidine kinase (TK) selection marker. A cassette containing IRES2-eGFP and a LoxP-flanked neomycin selection marker was inserted into an Nhel site between the translation stop codon (TGA) and the polyadenylation signal of the egr2 gene. ES cell clones from 129 mice were electroporated and selected for Neomycin resistance. ES cell clones were verified for homologous insertion in the endogenous locus by PCR and southern blot with 5′ and 3′ probes. Mice were backcrossed to C57BL/6 for over 8 generations.

TIL Isolation

Tumors were harvested from mice at the indicated time points. Tumors were dissociated through a 50 m filter and washed with PBS. TILs were further enriched by layering Ficoll-Hypaque beneath the cell suspension followed by centrifugation without breaks for 30 min at 400×g. The buffy-layer was isolated and washed twice with PBS before staining. For isolating specific cell populations by FACS, tumors were pooled when indicated and the cell layer was re-purified by Ficoll-Hypaque centrifugation twice. For day 28 tumors, after Ficoll-Hypaque separation, T cells were further purified by negative bead selection according to manufacturer's instructions (MAGNISORT, eBiosciences). Cells were then washed with PBS, stained at 4° C. for 15 minutes before resuspending in complete DMEM (cDMEM: 10% FBS, 100 U/mL Penicillin-Streptomycin, 1% MEM Non-Essential Amino Acids, 50 μM β-ME, 0.01M MOPS), and were sorted into either RLT lysis buffer (QIAGEN) or cDMEM depending on the experimental assay. Cells sorted into RLT buffer were put directly on dry ice as soon as the sort was finished.

Flow Cytometry and Antibodies

Cell suspensions were washed twice in PBS before staining an FACS buffer (10% FBS, 2 mM EDTA, 0.001% NaN₃). Cells were stained for 30 min on ice and fixed in 1% PFA. Antibodies against the following molecules were used: CD3 (17A2, AX700), 2B4 (2B4, FITC), CD127 (A7R34, PE), OX-40 (OX-86, PE), 4-1BB (17B5, Biotin, APC), CD160 (7H1, PE-Cy7), LAG-3 (C9B7W, PerCPeFluor710), PD-1 (RMP1-30, PE-Cy7), NRP1 (3E12, BV421), GITR (DTA-1, FITC), ICOS (7E.17G9, BV421), KLRG-1 (2F1, eF450, BV605), TIGIT (1G9, APC), TIM-3 (RMT3-23, PE), CD4 (RM4-5, BV605), CD45.1 (A20, FITC), CD45.2 (104, PE), CD8a (53-6.7, BV711). Fixable Viability Dye 506 (eBioscience) was used for live/dead discrimination. Staining of SIY-specific T cells was performed utilizing the SIYRYYGL-Pentamer (PE) (Proimmune); a SIINFEKL-pentamer (PE) was used as a non-specific control. All flow cytometric analysis was conducted on an LSRFortessa (BD) and analyzed using FlowJo software (Tree Star).

Quantitative Real-Time PCR

Total RNA was extracted from sorted cell populations using the RNEasy Micro Kit (QIAGEN) following the manufacturer's protocol. cDNA was synthesized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to manufacturer's instructions. Transcript levels were determined using primer-probe sets (Tables 1a and 1b) developed through the online ProbeFinder Software and the Universal Probe Library (Roche) with the exception of IL-2 (Mm00434256_m1) and 18S (Hs99999901_s1). To minimize batch effect, when possible, all samples probed for a gene were run on the same 96-well qRT-PCR plate. All primer-probe sets either contained a primer spanning an exon-exon boundary or primers spanning an intron. Expression levels of transcripts were normalized to 18S expression

TABLE 1a Primer Sequences # Wilson IMGT Sequence  0 Cβ1.1 TRBC1 CTCAAACAAGGAGACCTTGGGTGG  1 Vβ1 TRVB5 CAGACAGCTCCAAGCTACTTTTAC  2 Vβ2 TRVB1 ATGAGCCAGGGCAGAACCTTGTAC  3 Vβ3 TRVB26 GAAATTCAGTCCTCTGAGGCAGGA  4 Vβ4 TRVB2 CTAAAGCCTGATGACTCGGCCACA  5 Vβ5.1 TRVB12-2 CTTTGGAGCTAGAGGACTCTGCCG  6 Vβ5.2 TRVB12-1 CCTTGGAACTGGAGGACTCTGCTA  7 Vβ6 TRVB19 GCCCAGAAGAACGAGATGGCCGTT  8 Vβ7 TRVB29 GGATTCTGCTAAAACAAACCAGACATCTGT  9 Vβ8.1 TRVB13-3 GCTTCCCTTTCTCAGACAGCTGTA 10 Vβ8.2 TRVB13-2 GCTACCCCCTCTCAGACATCAGTG 11 Vβ8.3 TRVB13-3 GGCTTCTCCCTCTCAGACATCTT 12 Vβ9 TRVB17 CTCTCTCTACATTGGCTCTGCAGG 13 Vβ10 TRVB4 CTTCGAATCAAGTCTGTAGAGCCG 14 Vβ11 TRVB16 TGAAGATCCAGAGCAGCGGGCCCC 15 Vβ12 TRVB15 CCACTCTGAAGATTCAACCTACAGAACCC 16 Vβ13 TRVB14 CAAGATCCAGTCTGCAAAGCAGGG 17 Vβ14 TRVB31 GCACGGAGAAGCTGCTTCTCAGCC 18 Vβ15 TRVB20 GCATATCTTGAAGACAGAGGC 19 Vβ16 TRVB3 CTCTGAAAATCCAACCCACAGCACTGG 20 Vβ17 TRVB24 TCTGAAGAAGACGACTCAGCACTG 21 Vβ18 TRVB30 GCAAGGCCTGGAGACAGCAGTATC

TABLE 1b Primer/Probe Roche Gene Primer1 Primer2 Probe # Lag3 tgctttgggaagctccagt gctgcagggaagatggac  79 Tnfrsf9 ccggtcttaagcacagacct gaacggtactggcgtctgtc 108 Egr2 CTACCCGGTGGAAGACCTC AATGTTGATCATGCCATCTCC  60 Sema7a tcaatcggctgcaagatgt cgcagacagctgagtagttcc  15 Crtam AGATCCAACAACGAGGAGACA TCATGCAACGCTTAGACTGG  71 Ccl1 tcaccatgaaacccactgc agcagcagctattggagacc  71 Ngn caccctagectaacctcaacc tgaaaacctcctcccctctt  45 Arl3 ctggcagatccagtcctgtt acccagttcatgccatcct 100 Exph5 atgagggaggagagcggtat cagcttgttgtccaaatcgtc  67 Fhl2 agaaaaccatcatgccaggt acaggtgaagcaggtctcgt  74 Nrn1 atcctcgcggtgcaaata gcccttaaagactgcatcaca 108 Ptgfrn ccggggagatctcatcaaa tcgaaggccatgtcatctg  12 Rankl tgaagacacactacctgactcctg cccacaatgtgttgcagttc  88 Hif1a gctgctcactgtgaaggaagt tggggaatgcattttaccat   2 Egr3 caatctgtaccccgaggaga ccgatgtccatcacattctct  74 Tnfa ctgtagcccacgtcgtagc ttgagatccatgccgttg  25 Gzmb gctgctcactgtgaaggaagt tggggaatgcattttaccat   2 Ccl1 tcaccatgaaacccactgc agcagcagctattggagacc  71 Ccl22 tcttgctgtggcaattcaga gcagagggtgacggatgtag  74

In Vivo Proliferation Assay

In vivo proliferation was measured by a BrdU pulse 24 hours prior to flow cytometric analysis. Each mouse received 0.8 mg BrdU injected i.p. on day 12 after tumor inoculation. TILs were isolated and surface stain was performed as described above. Following surface staining, cells were fixed and permeabilized using the Foxp3 staining kit (BD), according to manufacturer's protocol, and incubated with 100 μl PBS/DNase solution (300 μg/ml) for 30 minutes at 37° C. Cells were washed and incubated for 30 minutes at room temperature with anti-BrdU (FITC, Bu20a) and then washed with and resuspended in PBS.

In Vitro Stimulation Assays

Tissue culture-treated 96-well round bottom plates were coated with anti-CD3F (1 μg/ml; 2C11) in DPBS overnight at 4° C. or for 2 hours at 37° C. Cells were sorted into cold cDMEM media and put on ice as soon as the sort was finished. Cells were then pelleted, resuspended in 50 μl cDMEM and incubated with soluble anti-CD28 (2 μg/ml; PV-1) for 10-12 hours for a final volume of 100 μl. After stimulation supernatants were removed for ELISA or bead-based immunoassay (LegendPlex), and cells were washed once with DPBS and resuspended in 15 μl of RNAlater Stabilization Solution (QIAGEN) or 300 μl of RLT buffer. Cells were stored at −80° C. until RNA isolation was performed.

Protein Quantification

Measurement of protein concentration was determined either by a standard ELISA or bead-based immunoassay (LEGENDplex, BioLegend). ELISAs were performed according to manufacturer's protocol (Ready-SET-Go ELISA; eBioscience) on supernatants from in vitro stimulations. Absorbance values were obtained at 450 nm using an Emax microplate reader (Molecular Devices) and IL-2 concentration was determined by standard curve. Protein concentration values were normalized to the number of sorted cells plated. LEGENDplex assays were performed according to manufacturer's protocols. IL-2 concentration (FIG. 4B) was confirmed by both methods in separate experiments with no significant difference in IL-2 concentration between the two methods.

Spectratype Analysis and Sequencing

Mice were injected with 2×10⁶ B16. SIY.dsRed tumor cells. 14 days later, tumors were harvested and specific CD8⁺ TIL subpopulations were sorted into RLT buffer (QIAGEN) and immediately frozen. cDNA was synthesized from sorted cell populations and CDR3 regions were amplified by PCR with 21 different Vβ-5′ primers paired with a FAM-Cβ1.1 primer (Table 1). Three Vβ PCR reactions did not reach significant amplification for analysis and were removed from the analysis. For sequencing, Cβ-Vβ PCR products were purified using the QIAquick PCR purification kit (QIAGEN) and sequenced at the University of Chicago Genomics Core Facility. Cβ-Vβ PCR products were analyzed by capillary electrophoresis at the University of Chicago Genomics core and CDR3 peaks were aligned using the Liz500 ladder. Spectratype graphs were displayed using the GeneiousR9 software (Kearse et al., 2012). To generate the frequency profile for each Vβ spectratype, the area under each peak was measured using peak studio (fodorlab.uncc.edu/software/peakstudio). The Hamming Distance (Currier and Robinson, 2001; incorporated by reference in its entirety) was calculated between each Vβ spectratype from each CD8⁺ spleen and TIL population within a given mouse. To determine significance between the HD from each comparison the HDs for each Vβ from mice were averaged and a One-Way ANOVA with Dunn's correction for multiple comparisons was performed.

TCR Transgenic T Cell Transfer Experiments

Cell suspensions were generated from spleens and lymph nodes from congenic 2C/Rag2^(−/−)/CD45.1/2 and/or P14/Rag2^(−/−)/CD45.2 mice and T cells were purified by CD8⁺ negative selection (Miltenyi Biotechnologies) over magnetic columns according to the manufacturer's protocol. TCR Transgenic (Tg) T cells were washed with PBS, resuspended at a concentration of 10×10⁶/ml and 1×10⁶ TCR Tg cells were adoptively transferred into CD45.1 tumor bearing mice by tail vein transfer in a volume of 0.1 mL. After indicated times, 2C T cells and corresponding host CD8⁺ T cells were sorted and stimulated as described above.

In Vitro Cytotoxicity Assay

Per individual experiment, 10 C57BL/6 mice were injected s.c. with 2×10⁶ B16. SIY cells on both left and right flanks. On day 14, all 20 tumors were pooled and dissociated using the Tumor Dissociation Kit (Miltenyi Biotec) following the manufacturer's protocol. Tumor cell suspensions were washed 3-5 times with PBS and TILs were enriched for by Ficoll-Hypaque gradient centrifugation. TILs were stained, sorted and put directly on ice. TILs were titrated and added directly to a 96-well plate containing 50,000 P815 mastocytoma cells and 1 μg/mL anti-CD3. For a positive control, OT-I cells were isolated from OT-I/Rag2^(−/−) mice and stimulated with plate-bound anti-CD3 (0.25 μg/mL), anti-CD28 (2 μg/mL) and 100 U/mL IL-2 for 2-3 days. For a negative control, P815 cells were cultured alone or cultured with naïve CD8⁺ T cells isolated from lymph nodes. After 12 hours of incubation, cells were stained for Thy1, CD45, CD8a, Fixable Viability Dye 450 (eBioscience) and/or propidium Iodide.

Gene Expression Analysis

Total RNA for the CD8⁺ TIL subpopulations was isolated following the manufacturer's protocol (RNEasy Micro Kit: QIAGEN) from sorted cells pooled from 10 mice. Samples were analyzed by the University of Chicago Genomics Facility using Illumina MouseRef8 microarray chips. Two experimental replicates were performed, and the results were log₂ transformed and averaged. Probe sets that revealed a 1.5-fold difference abs(log₂(ratio)>1.5)) relative to CD8⁺4-1BB⁻LAG-3⁻PD-1⁻ cells were identified and used for subsequent analysis. The microarray data are available in the Gene Expression Omnibus database (ncbi.nlm.nih.gov/gds) under accession number GSE79919. For cross-study comparisons, log 2-fold change values were extracted using the GEO2R online software from the hypofunctional CD8⁺ TIL data set, GSE79858 ((GSM2107353, GSM2107353 and GSM2107355) versus (GSM2107350, GSM2107351, GSM210732)) and the CD8⁺ T cell exhausted data set, GSE41870 ((GSM1026819, GSM1026820, GSM1026821) versus (GSM1026786, GSM1026787, GSM1026788, GSM1026789)). Upregulated genes showing a 2-fold difference were used for analysis. Multiple genes names with from the GEO2R extracted data were identified and matched to gene names from the Illumina data set. The rank-rank hypergeometric overlap (RRHO) analysis (Plaisier et al., 2010; incorporated by reference in its entirety) was conducted at systems.crump.ucla.edu/rankrank/index.php and the associated Bioconductor package “RRHO” (Rosenblatt and Stein, 2014; incorporated by reference in its entirety).

Gene Ontology Enrichment Analysis

In a pair-wise fashion, shared upregulated genes were used as the input for the ClueGO software with the Cytoscape application (Shannon et al., 2003; incorporated by reference in its entirety). Both the Biological Process and Immune System Process Gene Ontology Annotations were used for analysis. Only pathways with a Bonferroni step down correction p-value >0.01 were considered when generating pathway nodes. Non-redundant pathways with the greatest number of genes found within each node were used as examples in FIG. 6A.

Antibody and FTY720 Treatments

Mice were treated i.p. with 100 μg/mouse of anti-4-1BB (Bio-X-Cell; LOB12.3) antibody and/or 100 μg/mouse anti-LAG-3 (Bio-X-Cell; C9B7W). For tumor outgrowth experiments, mice were treated on day 7, 10, 13 and 16 after tumor inoculation. For ex vivo functional experiments mice were treated on day 7, 10 and 13 and cells were sorted on day 14. For experiments blocking lymph node egress, 25 μg of FTY720 was given by gavage one day prior to first antibody treatment (day 6) and continued every day until endpoint on day 14.

Results 4-1BB and LAG-3 Identify a Major Population of CD8⁺ TILs

To determine whether 4-1BB and LAG-3 could identify dysfunctional CD8⁺ TILs, the expression pattern of LAG-3 and 4-1BB was examined using the well-characterized B16. SIY model of melanoma. On day 7 following tumor inoculation, the 4-1BB⁺LAG-3⁺ population comprised 15.8% of all CD8⁺ TILs. The frequency of this population significantly increased to 44% by day 21. The frequency of 4-1BB⁻LAG-3⁺ (4⁻L⁺) population also increased 1.9-fold from day 7 to day 14 to comprise 25% of the CD8⁺ TIL compartment. In contrast, the frequency of the 4-1BB⁻LAG-3⁻ (4⁻L⁻) population decreased by 2.7-fold by day 21. There was no significant increase in the proportion or number of 4-1BB⁺LAG-3⁻ CD8⁺ TILs within the time frame of the experiment (FIGS. 1A and B). Similar patterns were seen when analyzing absolute numbers of cell subsets (FIGS. 1C and D). Acquisition of these phenotypes was specific for the tumor microenvironment, as they were not observed in the spleen or tumor-draining lymph node (TdLN) (FIG. 1A). These data indicate that the tumor microenvironment preferentially supports the induced co-expression of LAG-3 and 4-1BB.

The selective increase in cell numbers and proportional shift towards the 4-1BB⁻LAG-3⁺ and 4-1BB⁺LAG-3⁺ populations during tumor progression indicated that expansion of these populations was occurring within the tumor microenvironment. CD8⁺ TILs were stained for Ki67 at day 14 after tumor inoculation and analyzed by flow cytometry. 81% of 4-1BB⁻LAG-3⁺ cells and 85% of 4-1BB⁺LAG-3⁺ cells were Ki67⁺ compared to only 32% of the 4-1BB⁻LAG-3⁻ TILs (FIG. 1E). Mice were pulsed with BrdU on day 12, and 24 hours later the CD8⁺ TIL subpopulations were analyzed for BrdU incorporation. Indeed, the 4-1BB⁻LAG-3⁺ and 4-1BB⁺LAG-3⁺ populations incorporated more BrdU compared to the 4-1BB⁻LAG-3 population (FIG. 1F). These data indicate that once CD8⁺ T cells arrive at the tumor site, a fraction of TILs expands within the tumor, and that these expanding TILs are identified by increased expression of 4-1BB and LAG-3.

To determine if upregulation of LAG-3 and 4-1BB was simply a product of the B16. SIY tumor model or if it is a more general feature of CD8⁺ T cells within tumors, T cells from three additional progressively growing tumor models, C1498. SIY, MC38. SIY, EL4. SIY and B16F10 parental were analyzed. TILs were analyzed for expression of 4-1BB and LAG-3 at day 14. We found that the pattern of expression was similar to that seen in CD8⁺ TILs isolated from B16. SIY tumors (FIGS. 1G and I). The results from the B16F10 parental tumor confirm that presence of SIY is not required to see co-expression of 4-1BB and LAG-3. In order to determine whether the 4-1BB⁺LAG-3⁺ TIL subset was generated only in progressing tumors or also in tumors that were rejected, T cell phenotypes in the 1969. SIY and MC57. SIY fibrosarcoma tumor models we analyzed, which are more immunogenic and undergo spontaneous rejection. Distinctly fewer 4-1BB⁺LAG-3⁺ cells were found among the CD8⁺ TIL compartment in the 1969. SIY and MC57. SIY tumors (Figure H and I). Over time, co-expression of 4-1BB and LAG-3 was maintained in B16. SIY tumors but not MC57. SIY tumors (FIG. 1J). These data indicate that the acquisition of the LAG-3⁺4-1BB⁺ TIL phenotype preferentially occurs within the tumor microenvironment and only upon conditions of tumor progression rather than regression.

CD8⁺4-1BB⁺LAG-3⁺ TILs Express Egr2 and Multiple Egr2 Gene Targets

Experiments conducted during development of embodiments herein to determine whether Egr2 expression itself was also characteristic of T cells within the CD8⁺ TIL compartment; an Egr2-IRES-GFP (Egr2^(GFP)) knock-in reporter mouse was utilized. Approximately 14% of all CD8⁺ TILs were GFP⁺ on both day 7 and day 14 (FIG. 2A). To confirm that Egr2 is faithfully reported, CD8⁺ TILs expressing high and low levels of EGFP were sorted and screened for Egr2 and several Egr2 targets by qRT-PCR. The Egr2-GFP^(hi) population expressed greater levels of Egr2 and many Egr2-target genes previously defined using in vitro anergy models. These include Tnfrsf9, Lag3, Ngn, Sema7a, Crtam, Ccl1 and Nrn1 (FIG. 2B). Expression of 4-1BB and LAG-3 in the Egr2-GFP^(hi) CD8⁺ TILs was confirmed by flow cytometry. The majority of Egr2-GFP^(hi) cells expressed LAG-3 and/or 4-1BB. The Egr2GFP^(lo) cells also showed expression of 4-1BB and LAG-3 on a subpopulation at day 14 (FIG. 2C). This result indicates either that CD8⁺ TILs expressing Egr2 encompass only a subset of the TILs expressing LAG-3 and/or 4-1BB, or that Egr2 is transiently expressed and is subsequently downregulated after the induction of LAG-3 and 4-1BB.

Using Egr2 target genes from in vitro anergic CD4⁺ T cell clones (Zheng et al., 2013; incorporated by reference in its entirety), the Egr2-driven transcriptional program was examined in sorted 4-1BB⁻LAG-3⁻ and 4-1BB⁺LAG-3⁺ cells by qRT-PCR. Of the 43 Egr2 target genes examined, 10 showed detectably increased expression in 4-1BB⁺LAG-3⁺ population, while expression of a similar subset of genes was increased in the 4-1BB⁻LAG-3⁺ population (FIG. 2D). Collectively, these data demonstrate that Egr2 is expressed in a subpopulation of CD8⁺ TILs expressing LAG-3 and/or 4-1BB, and that a subset of known Egr2 targets was detected in these larger T cell populations as a whole.

It was next examined whether Egr2 was required for expression of LAG-3 and 4-1BB among CD8⁺ TIL in vivo. To this end Egr2^(flox/flox)×pLCK-CreERT2×ROSA-YFP mice were utilized, in which oral tamoxifen administration results in a fraction of the CD8⁺ T cells deleting Egr2 and expressing YFP (FIG. 2E). This allowed comparison of both Egr2-sufficient (YFP⁻) and Egr2-deficient (YFP⁺) CD8⁺ within the same tumor. To determine that Egr2 was in fact deleted from the YFP⁺ fraction, both YFP⁺ and YFP⁻ CD8⁺ TILs were sorted and Egr2 transcripts were measured directly ex vivo and upon ex vivo stimulation. The YFP⁺ CD8⁺ TILs expressed substantially less Egr2 transcripts compared to the YFP⁻ counterparts (FIG. 2E). To determine if Egr2 is required for 4-1BB and LAG-3 expression, CD8⁺ TILs were analyzed at day 7 and 14 after tumor inoculation and compared the YFP⁺ and YFP⁻ populations to mice not treated with tamoxifen. At day 7, the YFP⁺ fraction expressed less 4-1BB and LAG-3 compared to the YFP-population and the WT CD8⁺ TILs. However, expression of 4-1BB and LAG-3 was not significantly different at day 14 (FIG. 2F). This indicates that other transcriptional regulators compensate and contribute to the expression of LAG-3 and 4-1BB, especially at later time points.

Egr3 has been shown to have overlapping function with Egr2 (Safford et al., 2005; incorporated by reference in its entirety) and HIF1α can contribute to 4-1BB expression (Palazón et al., 2012). To investigate whether these transcription factors may compensate for 4-1BB and/or LAG-3 expression we sorted Egr2GFP^(hi) and Egr2GFP^(lo) CD8⁺ TILs expressing 4-1BB and LAG-3 on day 7 and analyzed expression of Egr3 and HIF1α by qRT-PCR. Egr3 and HIF1α were indeed expressed in both the Egr2GFP^(hi) and Egr2GFP^(lo) populations. It was confirmed differential expression of Egr2 and CCL1 to between the Egr2GFP^(hi) and Egr2GFP^(lo) populations to assure sort purity (FIG. 2G). Together, these data indicate that Egr2 contributes to upregulation of 4-1BB and LAG-3 expression at early time points, but that other transcriptional regulators compensate and drive expression of LAG-3 and 4-1BB as the T cell-tumor interaction progresses.

CD8⁺4-1BB⁺LAG-3⁺ TILs are Oligoclonal and Enriched for Tumor Antigen Specificity

Not all T cells in the tumor microenvironment are specific for tumor-associated antigens, as memory T cells specific for irrelevant antigens are often found among TIL, and non-specific T cell trafficking has been documented in vivo (Harlin et al., 2006; incorporated by reference in its entirety). Experiments conducted during development of embodiments herein to determine whether 4-1BB⁺LAG-3⁺ CD8⁺ TILs are tumor-antigen specific. LAG-3, 4-1BB and Egr2 are upregulated after TCR stimulation and experiments indicate that this population expands within the tumor microenvironment in situ. Three complementary techniques were employed. First, the CD8⁺ TILs were isolated based on LAG-3 and 4-1BB expression by cell sorting and performed TCRβ spectratype analysis. Compared to the 4-1BB⁻LAG-3 TILs and CD8⁺ splenocytes, the 4-1BB⁺LAG-3⁺ TILs had a non-Gaussian distribution and shared one or two dominant peaks (FIG. 3A). Analysis of several Vβs displaying one dominant peak revealed that Vβ7 contained a single CDR3β sequence shared between the 4-1BB⁻LAG-3⁺ and 4-1BB⁺LAG-3⁺ populations, indicating a clonal relationship (FIG. 3A). To measure the oligoclonality of the CDR30 repertoires the Hamming Distance (HD) was calculated for each Vβ between the CD8⁺ TIL subpopulations and the splenic CD8⁺ population within three separate mice (FIG. 8 ). By transforming each spectratype into area under the curve frequency profiles the Hamming Distance computes the changes in frequency and reports a value of comparison between 0 and 1, with 0 indicating a completely identical frequency profile and 1 signifying a completely discordant profile. As a control, the HD of the splenic CD8⁺ populations between different mice was calculated (FIG. 3B, black bar). Since the splenic CD8⁺ spectratypes are largely Gaussian this value represents the HD between two similar distributions. Analysis of the HD between the CD8⁺ TIL subpopulations revealed that the 4-1BB⁺LAG-3⁺ and 4-1BB⁻LAG-3⁺ but not the 4-1BB⁻LAG-3⁻ CDR30 distributions are significantly different (less Gaussian) compared to the splenic CD8⁺ population (FIG. 3B). These data indicate that the 4-1BB⁺LAG-3⁺ and 4-1BB-LAG-3⁺ populations are oligoclonal expanded subsets of TILs, indicating antigen specificity in these subpopulations.

As a second approach, the B16. SIY melanoma and MC38. SIY adenocarcinoma models were utilized. CD8⁺ T cells specific for the H-2K^(b)-restricted SIY epitope (SIYRYYGL) were monitored. SIYRYYGL/K^(b) pentamer⁺ (H-2K^(b)/SIY) cells were found in expanded numbers within B16. SIY and MC38. SIY tumors at day 14 after tumor inoculation (FIG. 3C). Nearly 47% of the H-2K^(b)/SIY⁺ cells expressed both 4-1BB and LAG-3, in contrast to 32% of the H-2K^(b)/SIY⁻ population (FIGS. 3C and E). This enrichment of antigen-specific CD8⁺ TILs in the 4-1BB⁺LAG-3⁺ populations indicates that these markers identify tumor antigen-specific TILs. The H-2K^(b)/SIY⁻ cells also contained significant numbers of 4-1BB⁺LAG-3⁺ cells, which is consistent with the notion that tumor antigens other than SIY are also recognized by subsets of CD8⁺ TILs in vivo (FIG. 3C). H-2K^(b)/SIY⁺ cells in the spleen or TdLN did not co-express 4-1BB and LAG-3, indicating that this phenotype is acquired within the tumor microenvironment.

These features were also analyzed t in the context of tumor-antigen specific CD8⁺ TILs in two spontaneously rejected tumor models. To this end, H-2K^(b)/SIY-specific CD8⁺ TILs cells were evaluated from MC57. SIY and 1969. SIY tumors. At day 14 after tumor inoculation, approximately 5% of the H-2K^(b)/SIY-specific CD8⁺ TILs were found in the 4-1BB⁺LAG-3⁺ fraction. As with the B16. SIY tumors, no H-2K^(b)/SIY-specific CD8 T cells co-expressed 4-1BB and LAG-3 in the TdLN or spleen (not shown) (FIG. 3D). Unlike the B16. SIY and MC38. SIY tumors, no significant enrichment of 4-1BB⁺LAG-3⁺ H-2K^(b)/SIY-specific CD8⁺ TILs was observed (FIGS. 3D and E). These data indicate that tumor antigen specificity per se does not determine dysfunctionality, and that this is a feature unique to the microenvironment of progressing tumors.

As a third measure to determine if tumor-antigen specific CD8⁺ T cells acquire the 4-1BB⁺LAG-3⁺ phenotype, congenically marked 2C and P14 transgenic (Tg) T cells, isolated from 2C/Rag2^(−/−) and P14/Rag2^(−/−) mice, were transferred into tumor-bearing hosts. The 2C TCR is specific for the SIY model antigen expressed by B16. SIY tumor cells, while P14 is an irrelevant TCR specific for the LCMV-derived gp33_41 epitope; both TCRs are H-2K^(b)-restricted. 2C and P14 Tg CD8⁺ T cells were transferred via tail vein 7 days after tumor inoculation. Seven days after transfer, tumors and TdLNs were extracted and the phenotypic profile of the transferred populations was analyzed. This system allowed for the analysis of two T cell populations with defined antigen specificities within the same tumor microenvironment, as well as the polyclonal host CD8⁺ T cells. The 2C T cells were more efficiently recruited and expanded within the tumor microenvironment compared to the P14 T cells and encompassed a large fraction of the total CD8⁺ TIL population (FIG. 3F). Of the 2C T cells, nearly all expressed LAG-3 and or 4-1BB while this was true for only a small percentage of the P14 cells (FIGS. 3G and H). Consistent with the SIY-K^(b) pentamer analysis, the co-expression of LAG-3 and 4-1BB on 2C T cells was not observed in the TdLN. Together, these results demonstrate that the 4-1BB⁺LAG-3⁺ phenotype is a property of tumor antigen-specific TIL under conditions of tumor progression.

CD8⁺ TILs Expressing LAG-3 and 4-1BB Exhibit Defective IL-2 Production Yet Produce IFN-γ and Treg-Recruiting Chemokines

Based on the characteristics of the in vitro T cell anergy model that led to the identification of Egr2 as an important regulator, experiments conducted during development of embodiments herein to determine whether the tumor-antigen specific 4-1BB⁺LAG-3⁺ CD8⁺ TIL population is dysfunctional in their capacity to produce IL-2. To this end each subpopulation was sorted and stimulated with anti-CD3 and anti-CD28 mAb and analyzed IL-2 production by qRT-PCR and ELISA. Since nearly all CD8⁺ TILs displayed an activated phenotype we used CD8⁺CD44⁺ splenocytes as a positive control. Indeed, the 4-1BB⁺LAG-3⁺ cells showed a 100-fold reduction in Il-2 mRNA and as much as a 40-fold reduction in IL-2 protein levels compared to the 4-1BB⁻LAG-3 population (FIGS. 4A and 4B). As a second approach, Egr2^(hi) TIL (which are also largely 4-1BB⁺LAG-3⁺) was examined by utilizing the Egr2-GFP reporter mice. Indeed, ex vivo stimulated Egr2-GFP^(hi) CD8⁺ TILs also exhibited reduced Il-2 transcript compared to Egr2-GFP^(lo) cells (FIG. 4C). As a final approach, congenically marked 2C T cells were adoptively transferred intravenously into tumor-bearing hosts and recovered the 2C T cells 7 days later from the tumor and TdLN. 2C T cells isolated from tumors exhibited a reduced capacity to produce Il-2 transcripts, at a level equivalent to 4-1BB⁺LAG-3⁺ TILs, compared to 2C CD44⁺ T cells isolated from the TdLN (FIG. 4D). In chronic infection models, expression of PD-1 has been suggested to identify intrinsically dysfunctional or “exhausted” CD8⁺ T cells. To determine if PD-1 alone might be sufficient to identify cells that lack the capacity to produce IL-2, CD8⁺ TILs that lacked expression of LAG-3 and 4-1BB were isolated and tested for the ability of the PD-1⁺ fraction to produce IL-2. Approximately ˜10% of CD8⁺ TILs were 4-1BB-LAG-3-PD-1⁺ on day 14 and 21 (FIGS. 4E and F). Upon ex vivo stimulation, this population retained the capacity to produce 11-2 mRNA at a level comparable to the 4-1BB⁻LAG-3⁻ cells (FIG. 4G). These results indicate that PD-1 expression alone is not sufficient to identify dysfunctional TIL in the tumor microenvironment.

To further examine functional alterations during tumor progression we tested for protein levels of IL-2, IFN-γ and TNF-α after TCR stimulation. As the loss of the ability of CD8⁺ TILs to produce cytokines is suggested to be a temporal process reported initiated following entry into the tumor microenvironment (Waugh et al., 2016; Schietinger et al., 2016; incorporated by reference in their entireties) or progressively after 30 days in the chronic LCMV model (Wherry et al., 2007; incorporated by reference in its entirety), cytokine production was tested on day 7, 14, 21 and 28. The 4-1BB⁺LAG-3⁺ population lost the capacity to produce IL-2 as early as day 7 while the 4-1BB⁻LAG-3⁺ population lost IL-2 production between day 7 and day 14 (FIG. 5A). The 4-1BB⁻LAG-3⁻ population did not lose the ability to produce IL-2 at any time point tested (FIG. 5A), supporting the notion that this population is not tumor antigen specific and that differentiation into the dysfunctional state is an antigen-dependent process (Schietinger et al., 2016; incorporated by reference in its entirety). The 4-1BB⁺LAG-3⁺ population produced more IFN-γ at all time points after day 7 compared to their negative counterparts, albeit with a slight decrease in IFN-γ production over time. While the increase in IFN-γ was maintained until later time points, TNF-α production was lost by day 28 (FIG. 5A).

Experiments were conducted during development of embodiments herein to evaluate production of cytokines directly in the tumor without in vitro restimulation, which may more closely reflect which T cells were receiving TCR stimulation in situ. Each T cell population was sorted directly ex vivo without any culturing and mRNA levels were measured by qRT-PCR. Elevated Ifn-γ and Gzmb transcripts were observed from the 4-1BB⁺LAG-3⁺ subpopulation, along with a slight decrease in Tnf-α levels, compared to the 4-1BB⁻LAG-3⁻ cells (FIG. 5B). Production of IFN-γ in primary TILs was confirmed by injecting tumors with Brefeldin A prior to analysis by intracellular cytokine staining. Consistent with the mRNA expression, the 4-1BB⁺LAG-3⁺ population produced significantly greater amounts of IFN-γ protein (FIG. 5C). Thus, the 4-1BB⁺LAG-3⁺ TIL are not completely devoid of functionality, as they continue to produce IFN-γ despite defective production of IL-2. This phenotype is consistent with in vitro T cell anergy models (Jenkins et al., 1987; incorporated by reference in its entirety).

To test whether the 4-1BB⁺LAG-3⁺ population still retains cytotoxic capacity, re-directed lysis was performed by co-culturing anti-CD3 bound P815 mastocytoma target cells with the different CD8⁺ TIL subpopulations directly after sorting. 4-1BB⁺LAG-3⁺CD8⁺ TILs isolated from day 14 tumors were able to lyse target cells at a comparable efficacy to in vitro primed OT-I cells. 4-1BB⁺LAG-3⁺ TILs isolated from day 21 tumors were still able to lyse target cells, albeit to a lesser extent compared to primed OT-I cells (FIG. 5D).

CD8⁺ T cells in the tumor can be the source of the chemokine CCL22 that recruits FoxP3⁺ regulatory T cells (Tregs) to the tumor microenvironment (Spranger et al., 2013; incorporated by reference in its entirety). In addition, the chemokine Ccl1 was an Egr2 target in anergic T cells (Zheng et al., 2013; incorporated by reference in its entirety), and it has been suggested that CCL1 also contribute to Treg recruitment in the tumor context in vivo (Hoelzinger et al., 2010; incorporated by reference in its entirety). However, whether all CD8⁺ T cells in the tumor produce these chemokines or if they are only produced by subpopulations of T cells had not been determined. To address this the CD8⁺ TIL phenotypic subpopulations were analyzed for Ccl1 and Ccl22 mRNA expression directly ex vivo by qRT-PCR. Indeed, the 4-1BB⁺LAG-3⁺TIL population produced substantially greater Ccl1 and Ccl22 compared to their negative counterparts or to splenic CD8⁺CD44⁺ T cells (FIG. 4K). As a control, expression of a distinct chemokine Ccl5 was found not to be differentially expressed.

Together, these data show that co-expression of 4-1BB and LAG-3 delineates tumor antigen-specific CD8⁺ TIL that lack the ability to produce IL-2 yet retain the ability to produce IFN-γ, kill target cells in vitro, and secrete chemokines capable of Treg recruitment. Given the fact that IFN-γ is responsible for the upregulation of PD-L1 and IDO in the tumor microenvironment, and that chemokines produced by CD8⁺ TIL contribute to Treg recruitment (Spranger et al., 2013; incorporated by reference in its entirety), these data indicate that the 4-1BB⁺LAG-3⁺ population contributes to the network of immune suppressive mechanisms within the tumor microenvironment that limit the efficacy of anti-tumor immunity.

Gene Expression Profiling Reveals that CD8⁺4-1BB⁺LAG-3⁺ TILs Express an Extensive Array of Additional Co-Stimulatory and Co-Inhibitory Receptors

Having in hand surface markers that define tumor antigen-specific dysfunctional CD8⁺ TILs, experiments conducted during development of embodiments herein to compare the gene expression profile of this population to other published profiles of dysfunctional CD8⁺ T cells to determine genes that regulate or are differentially expressed in cells in this dysfunctional state. To this end, a cross-study comparison was conducted of the transcriptional profiles of the “dysfunctional” 4-1BB⁺LAG-3⁺CD8⁺ TILs, “hypofunctional” CD8⁺ TILs from a study utilizing the murine CT26 tumor model (Waugh et al., 2016; incorporated by reference in its entirety) and LCMV “exhausted” GP33 specific CD8⁺ T cells (Doering et al., 2012; incorporated by reference in its entirety). The results are depicted in Table 2. Only genes with a 2-fold increase over controls from each study independently were considered. Over a 2-fold greater number of genes was found to be shared between the dysfunctional TIL dataset and the previously published hypofunctional CD8⁺ TIL data, than with the exhausted T cell profile (FIG. 6A). In addition, a rank-rank hypergeometric overlap (RRHO) analysis indicated a greater statistically significant overlap (FIG. 10A) and a greater correlation (FIG. 10B) between the current dysfunctional TIL and the published hypofunctional CD8⁺ TIL gene expression profiles compared to the virally-induced exhausted CD8⁺ T cell profile, indicating a more similar molecular program between CD8⁺ T cells isolate from tumors compared to chronic viral infection.

TABLE 2 Differentially regulated genes in CD8⁺ 4-1BB⁺ LAG-3⁺ TILs Log2-Fold Gene Log2-Fold Gene Gene Description Change Symbol Gene Description Change GLDC glycine decarboxylase 11.25109772 CRY2 cryptochrome circadian −1.546648257 clock 2 GZMD Granzyme D 10.66720027 KCMF1 potassium channel −1.546835341 modulatory factor 1 SLC17A6 solute carrier family 17 8.946467699 RHOB ras homolog family member −1.548813112 member 6 B IL1R2 interleukin 1 receptor type 7.595353131 KRT15 keratin 15 −1.549018071 2 LTF lactotransferrin 7.530211233 RRAD RRAD, Ras related −1.549530357 glycolysis inhibitor and calcium channel regulator NRGN neurogranin 7.334049768 C3 complement component 3 −1.549960037 GZME granzyme E 7.160375687 ITFG3 Description Not Found −1.550162812 RPL6 ribosomal protein L6 7.142107057 HAAO 3-hydroxyanthranilate 3,4- −1.550553207 dioxygenase NRN1 neuritin 1 7.087993146 RNF138 ring finger protein 138 −1.551449524 LPL lipoprotein lipase 7.004501392 UNC93B1 unc-93 homolog B1 −1.551767491 (C. elegans) CLGN calmegin 6.933690655 ANKZF1 ankyrin repeat and zinc −1.552214097 finger domain containing 1 CD70 CD70 molecule 6.906890596 IFITM3 interferon induced −1.552644542 transmembrane protein 3 AREG amphiregulin 6.712870868 TXNIP thioredoxin interacting −1.552785452 protein ZRANB3 zinc finger RANBP2-type 6.595443985 LMAN1L lectin, mannose binding 1 −1.554588852 containing 3 like ASNS asparagine synthetase 6.59496878 ALDH3B1 aldehyde dehydrogenase 3 −1.554711558 (glutamine-hydrolyzing) family member B1 FANCD2 Fanconi anemia 6.353146826 GIP gastric inhibitory −1.555511104 complementation group polypeptide D2 GM156 predicted gene 6.293701542 COX7A2L cytochrome c oxidase −1.555572553 156(Gm156) subunit 7A2 like ACAA1B acetyl-Coenzyme A 6.293701542 APPL2 adaptor protein, −1.555598704 acyltransferase phosphotyrosine interacting 1B(Acaa1b) with PH domain and leucine zipper 2 IGF2BP3 insulin like growth factor 6.186857067 KLHL22 kelch like family member −1.555929583 2 mRNA binding protein 22 3 GZMG granzyme G 6.093813673 OLFR272 olfactory receptor −1.557482156 272(Olfr272) CIB2 calcium and integrin 6.007868243 LRRC29 leucine rich repeat −1.559366716 binding family member 2 containing 29 ATG9B autophagy related 9B 5.986410935 A630095E13RIK Description Not Found −1.560714954 XKR8 XK related 8 5.977279924 OLFR194 olfactory receptor −1.560714954 194(Olfr194) EPDR1 ependymin related 1 5.956521363 OLFR1013 olfactory receptor −1.560714954 1013(Olfr1013) SPP1 secreted phosphoprotein 1 5.797769502 GLRA4 glycine receptor alpha 4 −1.560714954 RGS8 regulator of G-protein 5.753805672 P2RY6 pyrimidinergic receptor −1.560714954 signaling 8 P2Y6 MDFIC MyoD family inhibitor 5.730639956 RASGEF1B RasGEF domain family −1.560714954 domain containing member 1B DMWD dystrophia myotonica, 5.687200695 IL22RA2 interleukin 22 receptor −1.560714954 WD repeat containing subunit alpha 2 KIF11 kinesin family member 11 5.669593751 LIN7C lin-7 homolog C, crumbs −1.560714954 cell polarity complex component LGI2 leucine rich repeat LGI 5.655351829 DMRT1 doublesex and mab-3 −1.560714954 family member 2 related transcription factor 1 ZFP41 ZFP41 zinc finger protein 5.615445725 TSPAN12 tetraspanin 12 −1.560714954 MLKL mixed lineage kinase 5.605849867 PAK3 p21 (RAC1) activated −1.560714954 domain-like kinase 3 CENPH centromere protein H 5.563768278 COL2A1 collagen type II alpha 1 −1.560714954 chain SERPINF1 serpin family F member 1 5.5360529 SLC37A1 solute carrier family 37 −1.560714954 member 1 UNC13B unc-13 homolog B 5.503030646 PSD3 pleckstrin and Sec7 domain −1.560714954 (C. elegans) containing 3 MLANA melan-A 5.496654083 RDH5 retinol dehydrogenase 5 −1.560714954 PES1 pescadillo ribosomal 5.484376709 ABCA3 ATP binding cassette −1.561263453 biogenesis factor 1 subfamily A member 3 2900026A02RIK Description Not Found 5.477353527 PLA2G4E phospholipase A2 group −1.561650879 IVE OSR2 odd-skipped related 5.416164165 DDIT3 DNA damage inducible −1.563566526 transciption factor 2 transcript 3 MPP6 membrane palmitoylated 5.408506442 ZFP12 zinc finger protein −1.564308646 protein 6 12(Zfp12) HIST1H3C histone cluster 1, H3c 5.397460726 PIGYL phosphatidylinositol glycan −1.564585219 anchor biosynthesis, class Y-like(Pigyl) PI4K2B phosphatidylinositol 4- 5.375039431 CCDC97 coiled-coil domain −1.565355117 kinase type 2 beta containing 97 SH3YL1 SH3 and SYLF domain 5.375039431 OLFR1112 olfactory receptor −1.56589319 containing 1 1112(Olfr1112) RAD51 RAD51 recombinase 5.371558863 ACTN2 actinin alpha 2 −1.566931646 ZBTB32 zinc finger and BTB 5.318316841 POLG polymerase (DNA) gamma, −1.567265595 domain containing 32 catalytic subunit MSC musculin 5.285402219 FBXO32 F-box protein 32 −1.567281905 TG thyroglobulin 5.259272487 MRPL15 mitochondrial ribosomal −1.570722678 protein L15 RSPH1 radial spoke head 1 5.236492618 FCHSD2 FCH and double SH3 −1.571821211 homolog domains 2 ARL11 ADP ribosylation factor 5.21916852 RECQL RecQ like helicase −1.572889668 like GTPase 11 NUDT11 nudix hydrolase 11 5.215290306 NDUFB11 NADH:ubiquinone −1.572889668 oxidoreductase subunit B11 APBB1 amyloid beta precursor 5.197708158 SOX8 SRY-box 8 −1.573341535 protein binding family B member 1 SPINK2 serine peptidase inhibitor, 5.189824559 1700030J22RIK Description Not Found −1.57662394 Kazal type 2 HMGN3 high mobility group 5.168922782 EMB embigin −1.577890585 nucleosomal binding domain 3 FAM20B family with sequence 5.12722055 CELSR1 cadherin EGF LAG seven- −1.578201987 similarity 20 member B pass G-type receptor 1 CDC25C cell division cycle 25C 5.11997861 COL1A2 collagen type I alpha 2 −1.580682782 chain FAM20A family with sequence 5.108524457 1700080E11RIK Description Not Found −1.581046002 similarity 20 member A PPP1R16B protein phosphatase 1 5.09592442 GALNT12 polypeptide N- −1.581363645 regulatory subunit 16B acetylgalactosaminyl- transferase 12 SBNO1 strawberry notch homolog 5.050936965 RMND5B required for meiotic nuclear −1.583960816 1 (Drosophila) division 5 homolog B ST14 suppression of 5.026800059 LRRC28 leucine rich repeat −1.583987499 tumorigenicity 14 containing 28 LRRC49 leucine rich repeat 5.024704311 OLFR622 olfactory receptor −1.584962501 containing 49 622(Olfr622) TIAM1 T-cell lymphoma invasion 5.004501392 OLFR339 olfactory receptor −1.584962501 and metastasis 1 339(Olfr339) APLF aprataxin and PNKP like 4.951867504 NEIL3 nei like DNA glycosylase 3 −1.584962501 factor PGPEP1 pyroglutamyl-peptidase I 4.927185358 SNX24 sorting nexin 24 −1.584962501 ALCAM activated leukocyte cell 4.909293086 SLC7A11 solute carrier family 7 −1.584962501 adhesion molecule member 11 B9D1 B9 domain containing 1 4.906890596 FOXJ1 forkhead box J1 −1.584962501 SCIN scinderin 4.87282876 TAF3 TATA-box binding protein −1.584962501 associated factor 3 EXOC3L exocyst complex 4.844013973 MATN2 matrilin 2 −1.584962501 component 3-like(Exoc31) SLC35D3 solute carrier family 35 4.840463234 ADHFE1 alcohol dehydrogenase, iron −1.586280668 member D3 containing 1 ALDOC aldolase, fructose- 4.832890014 NANOS1 nanos C2HC-type zinc −1.586914831 bisphosphate C finger 1 TMEM205 transmembrane protein 4.830182468 PPP2R5B protein phosphatase 2 −1.586914831 205 regulatory subunit B′beta PLEKHA8 pleckstrin homology 4.820178962 USP22 ubiquitin specific peptidase −1.588703598 domain containing A8 22 SPC25 SPC25, NDC80 4.817623258 DAGLB diacylglycerol lipase beta −1.588817933 kinetochore complex component PCYT1B phosphate 4.749534268 KCTD6 potassium channel −1.589690033 cytidylyltransferase 1, tetramerization domain choline, beta containing 6 SLC6A8 solute carrier family 6 4.749534268 ACTL6B actin like 6B −1.591351555 member 8 TUBB6 tubulin beta 6 class V 4.749241128 FAM129B family with sequence −1.5915039 similarity 129 member B BSPRY B-box and SPRY domain 4.711494907 APOE apolipoprotein E −1.591683393 containing ICA1 islet cell autoantigen 1 4.708739041 GPR18 G protein-coupled receptor −1.592384168 18 TNFSF13B tumor necrosis factor 4.703211467 GSTP2 glutathione S-transferase, pi −1.592559885 superfamily member 13b 2(Gstp2) GSTCD glutathione S-transferase 4.700439718 GPR114 Description Not Found −1.593829527 C-terminal domain containing CCNB1 cyclin B1 4.699051844 CHUK conserved helix-loop-helix −1.594823937 ubiquitous kinase 4930539E08RIK Description Not Found 4.693211287 TAS1R3 taste 1 receptor member 3 −1.596595048 SRXN1 sulfiredoxin 1 4.66106548 SLC7A7 solute carrier family 7 −1.596935142 member 7 SERF1 small EDRK-rich factor 4.632268216 SPIB Spi-B transcription factor −1.597677703 1(Serf1) CCDC77 coiled-coil domain 4.62935662 POLR3A polymerase (RNA) III −1.599588488 containing 77 subunit A RHBDF1 rhomboid 5 homolog 1 4.626439137 OLFR952 olfactory receptor −1.599679175 952(Olfr952) REEP3 receptor accessory protein 4.599912842 1700021F05RIK Description Not Found −1.601623253 3 ITGA3 integrin subunit alpha 3 4.590961241 CCDC79 Description Not Found −1.602195565 SCCPDH saccharopine 4.590961241 FAM134B family with sequence −1.602715966 dehydrogenase (putative) similarity 134 member B MYADM myeloid associated 4.587964989 SEMA3B semaphorin 3B −1.602884409 differentiation marker FAM132A family with sequence 4.581953751 FA2H fatty acid 2-hydroxylase −1.604494406 similarity 132 member A FOXRED2 FAD dependent 4.572889668 ULK1 unc-51 like autophagy −1.604653903 oxidoreductase domain activating kinase 1 containing 2 CENPK centromere protein K 4.569855608 MCOLN1 mucolipin 1 −1.606242992 DCXR dicarbonyl and L-xylulose 4.562242424 BMP5 bone morphogenetic protein −1.606760033 reductase 5 TSPAN6 tetraspanin 6 4.54225805 ANKRD50 ankyrin repeat domain 50 −1.607137028 UPP1 uridine phosphorylase 1 4.53838296 OLFR560 olfactory receptor −1.608809243 560(Olfr560) DOK4 docking protein 4 4.520422249 OLFR366 olfactory receptor −1.608809243 366(Olfr366) ELOVL4 ELOVL fatty acid 4.501439145 OLFR273 olfactory receptor −1.608809243 elongase 4 273(Olfr273) KNDC1 kinase non-catalytic C- 4.499790117 FHIT fragile histidine triad −1.608809243 lobe domain containing 1 KRT17 keratin 17 4.491853096 AQP11 aquaporin 11 −1.608809243 CHST2 carbohydrate 4.487315031 TMEM176A transmembrane protein −1.608809243 sulfotransferase 2 176A TPX2 TPX2, microtubule 4.475733431 ENAH enabled homolog −1.608809243 nucleation factor (Drosophila) DUSP14 dual specificity 4.456149035 CLDN6 claudin 6 −1.608809243 phosphatase 14 BGN biglycan 4.449561375 SP1 Sp1 transcription factor −1.608809243 FKBP9 FK506 binding protein 9 4.442943496 SP140 SP140 nuclear body protein −1.608809243 CAPN5 calpain 5 4.385431037 RASGRP3 RAS guanyl releasing −1.608809243 protein 3 SLC1A4 solute carrier family 1 4.375039431 HIF3A hypoxia inducible factor 3 −1.609422664 member 4 alpha subunit IDI2 isopentenyl-diphosphate 4.357552005 FYCO1 FYVE and coiled-coil −1.611220598 delta isomerase 2 domain containing 1 AKR1E1 aldo-keto reductase 4.346596388 FBXL12 F-box and leucine rich −1.6119368 family 1, member repeat protein 12 E1(Akrle1) GNB4 G protein subunit beta 4 4.336088936 KLRA10 killer cell lectin-like −1.618484777 receptor subfamily A, member 10(Klra10) CPNE2 copine 2 4.318640898 ABAT 4-aminobutyrate −1.62058641 aminotransferase FAM132B family with sequence 4.259272487 AMHR2 anti-Mullerian hormone −1.62058641 similarity 132, member receptor type 2 B(Fam132b) SLC6A12 solute carrier family 6 4.259272487 DDX3Y DEAD-box helicase 3, −1.620649859 member 12 Y-linked CPLX1 complexin 1 4.240314329 LGALS4 galectin 4 −1.621550215 PDCD1 programmed cell death 1 4.221103725 SPG20 spastic paraplegia 20 −1.621653602 (Troyer syndrome) UTF1 undifferentiated 4.201633861 CTRL chymotrypsin like −1.62729369 embryonic cell transcription factor 1 WDR60 WD repeat domain 60 4.14974712 GREM2 gremlin 2, DAN family −1.627927342 BMP antagonist EGFL7 EGF like domain multiple 4.137503524 ZMAT3 zinc finger matrin-type 3 −1.628362075 7 ASPM abnormal spindle 4.133399125 AP4M1 adaptor related protein −1.628898157 microtubule assembly complex 4 mu 1 subunit TMBIM1 transmembrane BAX 4.104628811 NT5C2 5′-nucleotidase, cytosolic II −1.63059747 inhibitor motif containing 1 KNTC1 kinetochore associated 1 4.093952772 TMIE transmembrane inner ear −1.631606148 1700019D03RIK Description Not Found 4.087462841 OLFR556 olfactory receptor −1.632268216 556(Olfr556) TM4SF5 transmembrane 4 L six 4.087462841 OLFR463 olfactory receptor −1.632268216 family member 5 463(Olfr463) BIRC5 baculoviral IAP repeat 4.027905997 CTS3 cathepsin 3(Cts3) −1.632268216 containing 5 SYNGR3 synaptogyrin 3 4.022367813 OAS1B 2′-5′ oligoadenylate 1.632268216 synthetase 1B(Oas1b) PLSCR4 phospholipid scramblase 4 KCNF1 potassium voltage-gated −1.632268216 4 channel modifier subfamily F member 1 KIF15 kinesin family member 15 3.962376898 GCGR glucagon receptor −1.632268216 TICAM2 toll like receptor adaptor 3.958842675 NR1I3 nuclear receptor subfamily −1.632268216 molecule 2 1 group I member 3 CENPM centromere protein M 3.957682486 FSTL1 follistatin like 1 −1.632268216 KIF4 kinesin family member 3.956097191 ASAP3 ArfGAP with SH3 domain, −1.632268216 4(Kif4) ankyrin repeat and PH domain 3 E2F2 E2F transcription factor 2 3.93191939 IHH indian hedgehog −1.632268216 MSN moesin 3.930737338 SEMA3A semaphorin 3A −1.632268216 PTPRA protein tyrosine 3.928989949 RAMP1 receptor activity modifying −1.632575446 phosphatase, receptor protein 1 type A BC026585 cDNA sequence 3.882643049 NFKBID NFKB inhibitor delta −1.633158642 BC026585(BC026585) IQGAP3 IQ motif containing 3.867896464 KLK15 kallikrein related peptidase −1.633773522 GTPase activating protein 15 3 CD244 CD244 molecule 3.867896464 CYP1B1 cytochrome P450 family 1 −1.634684534 subfamily B member 1 HIST1H3G histone cluster 1, H3g 3.837943242 DNAJA1 DnaJ heat shock protein −1.635111002 family (Hsp40) member A1 SLC15A3 solute carrier family 15 3.832890014 SDSL serine dehydratase like −1.635807742 member 3 GIPC2 GIPC PDZ domain 3.817623258 CCDC137 coiled-coil domain −1.636838653 containing family containing 137 member 2 UTP15 UTP15, small subunit 3.812498225 ZSWIM4 zinc finger SWIM-type −1.638152805 processome component containing 4 PDIA6 protein disulfide 3.812498225 BBC3 BCL2 binding component 3 −1.638336813 isomerase family A member 6 JDP2 Jun dimerization protein 2 3.807354922 SOCS3 suppressor of cytokine −1.638876738 signaling 3 MESDC1 mesoderm development 3.806723946 2900092C05RIK Description Not Found −1.639157339 candidate 1 GAS2 growth arrest specific 2 3.802193217 CSRNP2 cysteine and serine rich −1.639383642 nuclear protein 2 IL4I1 interleukin 4 induced 1 3.802193217 BLOC1S3 biogenesis of lysosomal −1.639585785 organelles complex 1 subunit 3 PHF19 PHD finger protein 19 3.802193217 ELL elongation factor for RNA −1.64021945 polymerase II CKAP2L cytoskeleton associated 3.797012978 GTF3C4 general transcription factor −1.640658029 protein 2 like IIIC subunit 4 GSTT1 glutathione S-transferase 3.791814071 MYLPF myosin light chain, −1.640660074 theta 1 phosphorylatable, fast skeletal muscle ADAM3 a disintegrin and 3.781359714 CYP2A12 cytochrome P450, family 2, −1.641947141 metallopeptidase domain subfamily a, polypeptide 3 (cyritestin)(Adam3) 12(Cyp2a12) SLAMF7 SLAM family member 7 3.781359714 RNF139 ring finger protein 139 −1.642010395 MCPT8 mast cell protease 3.770829046 C78339 Description Not Found −1.643573868 8(Mcpt8) DGKG diacylglycerol kinase 3.765534746 EDEM1 ER degradation enhancing −1.64385619 gamma alpha-mannosidase like protein 1 NLGN2 neuroligin 2 3.716990894 UBE2E1 ubiquitin conjugating −1.645859791 enzyme E2 E1 SERPINE2 serpin family E member 2 3.694880193 PALMD palmdelphin −1.646322067 IL10 interleukin 10 3.689299161 AMICA1 adhesion molecule, interacts −1.647478619 with CXADR antigen 1(Amica1) SLC6A13 solute carrier family 6 3.689299161 KLHL11 kelch like family member −1.650611828 member 13 11 STAU2 staufen double-stranded 3.666756592 IFNGR2 interferon gamma receptor −1.651050175 RNA binding protein 2 2 (interferon gamma transducer 1) ARHGDIG Rho GDP dissociation 3.655351829 DECR1 2,4-dienoyl-CoA reductase −1.651406438 inhibitor gamma 1, mitochondrial TK1 thymidine kinase 1 3.637477097 SAMD3 sterile alpha motif domain −1.653213853 containing 3 PCYT1A phosphate 3.617728231 9130409123RIK Description Not Found −1.655351829 cytidylyltransferase 1, choline, alpha LAMB3 laminin subunit beta 3 3.608809243 2010107G12RIK Description Not Found −1.655351829 UBE2N ubiquitin conjugating 3.590961241 ZFP354B zinc finger protein −1.655351829 enzyme E2 N 354B(Zfp354b) STARD8 StAR related lipid transfer 3.578938713 TAS2R143 taste receptor, type 2, −1.655351829 domain containing 8 member 143(Tas2r143) PRR5 proline rich 5 3.578938713 OLFR65 olfactory receptor −1.655351829 65(Olfr65) BDH2 3-hydroxybutyrate 3.554588852 NRP neural regeneration −1.655351829 dehydrogenase, type 2 protein(Nrp) FAM124B family with sequence 3.548436625 DOK3 docking protein 3 −1.655351829 similarity 124 member B MGAT3 mannosyl (beta-1,4-)- 3.548436625 HIGD1A HIG1 hypoxia inducible −1.655351829 glycoprotein beta-1,4-N- domain family member 1A acetylglucosaminyl- transferase LAG3 lymphocyte activating 3 3.542346309 CCDC13 coiled-coil domain −1.655351829 containing 13 GDPD5 glycerophosphodiester 3.538812733 ANGPTL2 angiopoietin like 2 −1.655351829 phosphodiesterase domain containing 5 RNF168 ring finger protein 168 3.5360529 CNGB3 cyclic nucleotide gated −1.655351829 channel beta 3 LYPLA1 lysophospholipase I 3.529820947 HOXD4 homeobox D4 −1.655351829 TUBGCP4 tubulin gamma complex 3.523561956 KIFC3 kinesin family member C3 −1.655351829 associated protein 4 PYGL phosphorylase, glycogen, 3.51412226 AMACR alpha-methylacyl-CoA −1.655351829 liver racemase CCL3 C-C motif chemokine 3.510281539 2310014L17RIK Description Not Found −1.655707015 ligand 3 BCAT1 branched chain amino 3.508163667 BRAP BRCA1 associated protein −1.657090723 acid transaminase 1 ATP6V0A1 ATPase H+ transporting 3.501439145 SLC39A1 solute carrier family 39 −1.657631089 V0 subunit a1 member 1 EIF4E eukaryotic translation 3.498250868 OLFR419 olfactory receptor −1.65813796 initiation factor 4E 419(Olfr419) HIST1H4B histone cluster 1, H4b 3.491853096 NHP2L1 NHP2 non-histone −1.658298045 chromosome protein 2-like 1 (S. cerevisiae)(Nhp211) LAD1 ladinin 1 3.49085426 STOML2 stomatin like 2 −1.659357735 ITGAV integrin subunit alpha V 3.485426827 SAMM50 SAMM50 sorting and −1.662400762 assembly machinery component MRPL47 mitochondrial ribosomal 3.485426827 CCDC91 coiled-coil domain −1.6632299 protein L47 containing 91 CAMK2N1 calcium/calmodulin 3.484460783 ATF3 activating transcription −1.663483642 dependent protein kinase factor 3 II inhibitor 1 UEVLD UEV and lactate/malate 3.465974465 RAI1 retinoic acid induced 1 −1.663885989 dehyrogenase domains SFXN4 sideroflexin 4 3.462706751 RRAS2 related RAS viral (r-ras) −1.665826896 oncogene homolog 2 2810417H13RIK Description Not Found 3.461634298 UROS uroporphyrinogen III −1.665923156 synthase RAD51AP1 RAD51 associated protein 3.459431619 SCOC short coiled-coil protein −1.666272349 FUT4 fucosyltransferase 4 3.452858965 DUSP10 dual specificity phosphatase −1.666485948 10 CTNNBIP1 catenin beta interacting 3.44625623 CYB5R4 cytochrome b5 reductase 4 −1.666756592 protein 1 ZBTB8OS zinc finger and BTB 3.426264755 9930104L06RIK Description Not Found −1.667150978 domain containing 8 opposite strand LYSMD4 LysM domain containing 3.42259008 ZFP579 zinc finger protein −1.669023741 4 579(Zfp579) DIAP3 Description Not Found 3.40599236 RGP1 RGP1 homolog, RAB6A −1.669393721 GEF complex partner 1 PTGIS prostaglandin I2 3.399171094 PIAS2 protein inhibitor of −1.672137196 (prostacyclin) synthase activated STAT 2 MOAP1 modulator of apoptosis 1 3.392317423 METTL1 methyltransferase like 1 −1.672425342 SLC27A3 solute carrier family 27 3.392317423 POU5F1 POU class 5 homeobox 1 −1.673854965 member 3 MRPL39 mitochondrial ribosomal 3.371492175 SERPINB6C serine (or cysteine) −1.673932658 protein L39 peptidase inhibitor, clade B, member 6c(Serpinb6c) WTAP Wilms tumor 1 associated 3.364572432 STXBP4 syntaxin binding protein 4 −1.675552278 protein RAD54L RAD54-like 3.356589854 RIMS3 regulating synaptic −1.676120648 (S. cerevisiae) membrane exocytosis 3 CETN4 centrin 4(Cetn4) 3.336283388 XYLT2 xylosyltransferase 2 −1.676976793 CEP55 centrosomal protein 55 3.329123596 TAS2R107 taste receptor, type 2, −1.678071905 member 107(Tas2r107) CYP4F39 cytochrome P450, family 3.321928095 SKP1A S-phase kinase-associated −1.678071905 4, subfamily f, protein 1A(Skp1a) polypeptide 39(Cyp4f39) PTPN5 protein tyrosine 3.314696526 OLFR165 olfactory receptor −1.678071905 phosphatase, non-receptor 165(Olfr165) type 5 TUBE1 tubulin epsilon 1 3.292781749 OLFR111 olfactory receptor −1.678071905 111(Olfr111) TCAM1 testicular cell adhesion 3.285402219 CYP4A12A cytochrome P450, family 4, −1.678071905 molecule 1(Tcam1) subfamily a, polypeptide 12a(Cyp4a12a) MID1IP1 MID1 interacting protein 3.263034406 TLR6 toll like receptor 6 −1.678071905 1 ABHD6 abhydrolase domain 3.260682276 KCNS3 potassium voltage-gated −1.678071905 containing 6 channel modifier subfamily S member 3 ZCCHC4 zinc finger CCHC-type 3.255500733 FARSA phenylalanyl-tRNA −1.678071905 containing 4 synthetase alpha subunit MGST3 microsomal glutathione 3.25353624 SLC2A4 solute carrier family 2 −1.678071905 S-transferase 3 member 4 BC022687 cDNA sequence 3.247927513 GDPD4 glycerophosphodiester −1.678071905 BC022687(BC022687) phosphodiesterase domain containing 4 ACSF3 acyl-CoA synthetase 3.24325855 RCAN1 regulator of calcineurin 1 −1.678071905 family member 3 ADAM8 ADAM metallopeptidase 3.240314329 CCDC82 coiled-coil domain −1.678071905 domain 8 containing 82 SGCB sarcoglycan beta 3.237034772 CDYL2 chromodomain protein, Y- −1.678071905 like 2 SOCS2 suppressor of cytokine 3.232660757 MBD5 methyl-CpG binding −1.678071905 signaling 2 domain protein 5 HIST1H2AG histone cluster 1, H2ag 3.223000387 ACSL1 acyl-CoA synthetase long- −1.678071905 chain family member 1 CRMP1 collapsin response 3.201633861 OTUB2 OTU deubiquitinase, −1.678071905 mediator protein 1 ubiquitin aldehyde binding 2 RPS19BP1 ribosomal protein S19 3.201633861 NPPA natriuretic peptide A −1.678071905 binding protein 1 1700020L24RIK Description Not Found 3.193771743 LY96 lymphocyte antigen 96 −1.679594789 CCDC109B coiled-coil domain 3.181276986 OLFR351 olfactory receptor −1.680730557 containing 351(Olfr351) 109B(Ccdc109b) UBE2C ubiquitin conjugating 3.177917792 TGFBR1 transforming growth factor −1.681068055 enzyme E2 C beta receptor 1 SLC25A16 solute carrier family 25 3.177917792 KLHL6 kelch like family member 6 −1.683531539 member 16 ARHGAP19 Rho GTPase activating 3.167705534 ELMO2 engulfment and cell motility −1.683696454 protein 19 2 TYMS-PS thymidylate synthase, 3.166362514 POLR3D polymerase (RNA) III −1.683942043 pseudogene(Tyms-ps) subunit D IL3RA interleukin 3 receptor 3.145793675 RALGPS1 Ral GEF with PH domain −1.685524532 subunit alpha and SH3 binding motif 1 TMEM53 transmembrane protein 53 3.141596278 ATL2 atlastin GTPase 2 −1.685731341 THNSL2 threonine synthase like 2 3.141596278 RAD52 RAD52 homolog, DNA −1.689523672 repair protein 2810408M09RIK Description Not Found 3.129283017 GPC1 glypican 1 −1.689646894 ADAMDEC1 ADAM like decysin 1 3.121015401 ARHGAP15 Rho GTPase activating −1.690804518 protein 15 ASB2 ankyrin repeat and SOCS 3.118792343 GPRC5B G protein-coupled receptor −1.693999744 box containing 2 class C group 5 member B SLC37A4 solute carrier family 37 3.112700133 ZBTB1 zinc finger and BTB −1.694046727 member 4 domain containing 1 NICN1 nicolin 1 3.108478268 NARFL nuclear prelamin A −1.694880193 recognition factor like 2310067B10RIK Description Not Found 3.087462841 SLC26A6 solute carrier family 26 −1.695252347 member 6 PIGL phosphatidylinositol 3.077239787 MAPKBP1 mitogen-activated protein −1.695908738 glycan anchor kinase binding protein 1 biosynthesis class L 1190005I06RIK Description Not Found 3.070389328 RAB6B RAB6B, member RAS −1.697541036 oncogene family DHFR dihydrofolate reductase 3.070389328 ARL2 ADP ribosylation factor like −1.700349879 GTPase 2 FABP5 fatty acid binding protein 3.06608919 ZFP646 zinc finger protein −1.700439718 5 646(Zfp646) POMT2 protein O- 3.055794286 SELENBP2 selenium binding protein −1.700439718 mannosyltransferase 2 2(Selenbp2) F2RL2 coagulation factor II 3.053111336 ACOT3 acyl-CoA thioesterase −1.700439718 thrombin receptor like 2 3(Acot3) GRB7 growth factor receptor 3.048852907 REG3G regenerating family −1.700439718 bound protein 7 member 3 gamma SNX21 sorting nexin family 3.044394119 GAB1 GRB2 associated binding −1.700439718 member 21 protein 1 SUFU SUFU negative regulator 3.044394119 LCN10 lipocalin 10 −1.700439718 of hedgehog signaling RFC3 replication factor C 3.029288361 MTHFD2L methylenetetrahydrofolate −1.700439718 subunit 3 dehydrogenase (NADP+ dependent) 2-like CLDN12 claudin 12 3.017921908 PTCD3 pentatricopeptide repeat −1.700439718 domain 3 C1QTNF6 C1q and tumor necrosis 3.014450679 NTHL1 nth-like DNA glycosylase 1 −1.700439718 factor related protein 6 PLCXD1 phosphatidylinositol 2.99095486 NUDT3 nudix hydrolase 3 −1.700439718 specific phospholipase C X domain containing 1 SULT4A1 sulfotransferase family 2.99095486 CLEC12A C-type lectin domain family −1.700439718 4A member 1 12 member A CTTNBP2NL CTTNBP2 N-terminal 2.981852653 ZBTB3 zinc finger and BTB −1.700439718 like domain containing 3 SNX5 sorting nexin 5 2.977279924 AMT aminomethyltransferase −1.700439718 HPS5 HPS5, biogenesis of 2.972692654 ZDHHC14 zinc finger DHHC-type −1.700439718 lysosomal organelles containing 14 complex 2 subunit 2 WISP1 WNT1 inducible 2.968090752 NKX2-5 NK2 homeobox 5 −1.700491519 signaling pathway protein 1 PTPN9 protein tyrosine 2.963474124 FOXA3 forkhead box A3 −1.702815694 phosphatase, non-receptor type 9 USP37 ubiquitin specific 2.95419631 WASF1 WAS protein family −1.706412734 peptidase 37 member 1 SH3BGRL SH3 domain binding 2.935459748 OLFR690 olfactory receptor −1.707192688 glutamate rich protein like 690(Olfr690) NCALD neurocalcin delta 2.935459748 ENTPD5 ectonucleoside triphosphate −1.707764551 diphosphohydrolase 5 CDC42EP4 CDC42 effector protein 4 2.916476644 PCDHGA4 protocadherin gamma −1.709042655 subfamily A, 4 IGFBP7 insulin like growth factor 2.910553168 TCF12 transcription factor 12 −1.710308209 binding protein 7 ABHD4 abhydrolase domain 2.908868748 MTRR 5-methyltetrahydrofolate- −1.711494907 containing 4 homocysteine methyltransferase reductase CSF1 colony stimulating factor 2.906890596 CDKN1C cyclin dependent kinase −1.711690028 1 inhibitor 1C COX7A1 cytochrome c oxidase 2.897240426 PRICKLE1 prickle planar cell polarity −1.713410822 subunit 7A1 protein 1 TTYH2 tweety family member 2 2.892391026 ATXN7L1 ataxin 7 like 1 −1.71669984 ACO1 aconitase 1 2.87774425 SLCO3A1 solute carrier organic anion −1.719235762 transporter family member 3A1 BARD1 BRCA1 associated RING 2.867896464 TMEM110 transmembrane protein 110 −1.720046704 domain 1 GPN1 GPN-loop GTPase 1 2.867896464 KLF2 Kruppel like factor 2 −1.721374729 PTTG1 pituitary tumor- 2.867896464 FGG fibrinogen gamma chain −1.722466024 transforming 1 2810408A11RIK Description Not Found 2.857980995 ASAH2 N-acylsphingosine −1.722466024 amidohydrolase 2 BBX BBX, HMG-box 2.857980995 LAP3 leucine aminopeptidase 3 −1.722466024 containing LTBP3 latent transforming 2.837943242 STAB2 stabilin 2 −1.722466024 growth factor beta binding protein 3 ACTG2 actin, gamma 2, smooth 2.827819025 IL22RA1 interleukin 22 receptor −1.722466024 muscle, enteric subunit alpha 1 ISLR immunoglobulin 2.827819025 SERINC4 serine incorporator 4 −1.722466024 superfamily containing leucine rich repeat NARS2 asparaginyl-tRNA 2.823087408 GPR180 G protein-coupled receptor −1.722466024 synthetase 2, 180 mitochondrial (putative) ICAM4 intercellular adhesion 2.81452379 TIPARP TCDD inducible −1.722466024 molecule 4 (Landsteiner- poly(ADP-ribose) Wiener blood group) polymerase ABCB8 ATP binding cassette 2.813358991 USP11 ubiquitin specific peptidase −1.722466024 subfamily B member 8 11 IDI1 isopentenyl-diphosphate 2.811782922 TRIP6 thyroid hormone receptor −1.722466024 delta isomerase 1 interactor 6 GLS2 glutaminase 2 2.797012978 KCNH2 potassium voltage-gated −1.722466024 channel subfamily H member 2 HDAC8 histone deacetylase 8 2.797012978 ESR2 estrogen receptor 2 −1.722466024 BRIP1 BRCA1 interacting 2.797012978 FGF13 fibroblast growth factor 13 −1.722639247 protein C-terminal helicase 1 USP6NL USP6 N-terminal like 2.794415866 KBTBD7 kelch repeat and BTB −1.724237927 domain containing 7 TLCD2 TLC domain containing 2 2.791814071 UHRF1BP1 UHRF1 binding protein 1 −1.725835292 GUCY1A3 guanylate cyclase 1 2.787502763 BCAM basal cell adhesion −1.726509704 soluble subunit alpha molecule (Lutheran blood group) OCA2 OCA2 melanosomal 2.786596362 ELOVL6 ELOVL fatty acid elongase −1.726565554 transmembrane protein 6 VAT1 vesicle amine transport 1 2.772502543 PPM1K protein phosphatase, −1.726643643 Mg2+/Mn2+ dependent 1K HIST1H2AB histone cluster 1, H2ab 2.767914142 SPATA6 spermatogenesis associated −1.727673077 6 PIGC phosphatidylinositol 2.760220946 NAV1 neuron navigator 1 −1.727920455 glycan anchor biosynthesis class C PARG poly(ADP-ribose) 2.756558208 ANK3 ankyrin 3, node of Ranvier −1.727920455 glycohydrolase (ankyrin G) ESCO2 establishment of sister 2.754887502 KCNAB1 potassium voltage-gated −1.727920455 chromatid cohesion N- channel subfamily A acetyltransferase 2 member regulatory beta subunit 1 HIPK2 homeodomain interacting 2.754887502 CYP27A1 cytochrome P450 family 27 −1.727920455 protein kinase 2 subfamily A member 1 IMPA1 inositol monophosphatase 2.752945007 MAP4K4 mitogen-activated protein −1.729756006 1 kinase kinase kinase kinase 4 COQ4 coenzyme Q4 2.744161096 ANKRD7 ankyrin repeat domain 7 −1.730646873 ZBTB7A zinc finger and BTB 2.744161096 IFRD1 interferon related −1.732447522 domain containing 7A developmental regulator 1 GAMT guanidinoacetate N- 2.744161096 ALX3 ALX homeobox 3 −1.733354341 methyltransferase BIK BCL2 interacting killer 2.744161096 SNURF SNRPN upstream reading −1.733354341 frame PMS1 PMS1 homolog 1, 2.733354341 AMZ2 archaelysin family −1.73350053 mismatch repair system metallopeptidase 2 component HAVCR2 hepatitis A virus cellular 2.729769667 ROGDI rogdi homolog −1.73419198 receptor 2 FHL2 four and a half LIM 2.727254747 DAGLA diacylglycerol lipase alpha −1.734471203 domains 2 CHAF1A chromatin assembly 2.725248783 4930432K21RIK Description Not Found −1.736243886 factor 1 subunit A 2810004N23RIK Description Not Found 2.722466024 KRCC1 lysine rich coiled-coil 1 −1.73665741 TBC1D14 TBC1 domain family 2.722466024 OLFR1331 olfactory receptor −1.736826447 member 14 1331(Olfr1331) EHD2 EH domain containing 2 2.711494907 SLC25A25 solute carrier family 25 −1.73690749 member 25 APH1A aph-1 homolog A, 2.705977902 CXCR4 C-X-C motif chemokine −1.737779353 gamma-secretase subunit receptor 4 TMEM2 transmembrane protein 2 2.703211467 EPB4.1L3 Description Not Found −1.738767837 LCAT lecithin-cholesterol 2.700439718 CEP164 centrosomal protein 164 −1.738795736 acyltransferase FBXO15 F-box protein 15 2.689299161 AGER advanced glycosylation end −1.73961488 product-specific receptor ADAP1 ArfGAP with dual PH 2.674391397 B3GALT5 beta-1,3- −1.740215306 domains 1 galactosyltransferase 5 PPAPDC1B Description Not Found 2.666756592 OLFR450 olfactory receptor −1.74228265 450(Olfr450) CD48 CD48 molecule 2.666756592 ZFP780B zinc finger protein −1.744161096 780B(Zfp780b) CAMK4 calcium/calmodulin 2.655351829 OLFR485 olfactory receptor −1.744161096 dependent protein kinase 485(Olfr485) IV SAC3D1 SAC3 domain containing 2.64385619 OLFR47 olfactory receptor −1.744161096 1 47(Olfr47) ECHDC2 enoyl-CoA hydratase 2.640725033 CYP4F18 cytochrome P450, family 4, −1.744161096 domain containing 2 subfamily f, polypeptide 18(Cyp4f18) INCENP inner centromere protein 2.638460117 PLOD2 procollagen-lysine,2- −1.744161096 oxoglutarate 5-dioxygenase 2 INTS9 integrator complex 2.634920268 OSBPL1A oxysterol binding protein −1.744161096 subunit 9 like 1A KLRA17 killer cell lectin-like 2.632268216 CHRNA5 cholinergic receptor −1.744161096 receptor, subfamily A, nicotinic alpha 5 subunit member 17(Klra17) MAN2B2 mannosidase alpha class 2.632268216 TSSK4 testis specific serine kinase −1.744161096 2B member 2 4 DOLK dolichol kinase 2.632268216 ALKBH8 alkB homolog 8, tRNA −1.744161096 methyltransferase SAP30BP SAP30 binding protein 2.632268216 GPX2 glutathione peroxidase 2 −1.744161096 RTN1 reticulon 1 2.627898616 ATG4D autophagy related 4D −1.744161096 cysteine peptidase ADAM15 ADAM metallopeptidase 2.626439137 SCRN3 secernin 3 −1.744161096 domain 15 STAG3 stromal antigen 3 2.62058641 NOTCH3 notch 3 −1.744161096 NUDT2 nudix hydrolase 2 2.610775705 OLFR113 olfactory receptor −1.744357436 113(Olfr113) GLT8D2 glycosyltransferase 8 2.609988757 CD28 CD28 molecule −1.744605653 domain containing 2 CAPSL calcyphosine like 2.608809243 SAG S-antigen; retina and pineal −1.745224161 gland (arrestin) CALR calreticulin 2.608809243 AGTRAP angiotensin II receptor −1.749107415 associated protein CRYBG3 crystallin beta-gamma 2.605393551 BLK BLK proto-oncogene, Src −1.749534268 domain containing 3 family tyrosine kinase DIXDC1 DIX domain containing 1 2.596940379 MGAT5 mannosyl (alpha-1,6-)- −1.749534268 glycoprotein beta-1,6-N- acetyl- glucosaminyltransferase TACSTD2 tumor-associated calcium 2.593926161 RNF2 ring finger protein 2 −1.750890228 signal transducer 2 TRP53RK Description Not Found 2.588066506 COL14A1 collagen type XIV alpha 1 −1.752093722 chain PDCD1LG2 programmed cell death 1 2.584962501 PLEKHG3 pleckstrin homology and −1.752109698 ligand 2 RhoGEF domain containing G3 SEC23IP SEC23 interacting protein 2.584962501 ARHGEF18 Rho/Rac guanine nucleotide −1.754100479 exchange factor 18 ORM1 orosomucoid 1 2.584962501 LEF1 lymphoid enhancer binding −1.754887502 factor 1 ZFP322A zinc finger protein 2.575024164 COMMD9 COMM domain containing −1.75490709 322A(Zfp322a) 9 4931406C07RIK Description Not Found 2.560714954 SLC20A1 solute carrier family 20 −1.758637847 member 1 ZFP382 zinc finger protein 2.560714954 ACTR5 ARP5 actin-related protein −1.759244091 382(Zfp382) 5 homolog CLIP2 CAP-Gly domain 2.560714954 UBQLN3 ubiquilin 3 −1.765109548 containing linker protein 2 TNFAIP8L1 TNF alpha induced 2.560714954 ZFP770 zinc finger protein −1.765534746 protein 8 like 1 770(Zfp770) NRCAM neuronal cell adhesion 2.560714954 PCDHB18 protocadherin beta −1.765534746 molecule 18(Pcdhb18) HPSE heparanase 2.560714954 OLFR700 olfactory receptor −1.765534746 700(Olfr700) RTKN rhotekin 2.558985655 FOXP4 forkhead box P4 −1.765534746 DLGAP5 DLG associated protein 5 2.550125328 CDC34 cell division cycle 34 −1.765534746 ENPP2 ectonucleotide 2.548436625 HIST1H1E histone cluster 1, H1e −1.765534746 pyrophosphatase/ phosphodiesterase 2 GCNT1 glucosaminyl (N-acetyl) 2.548436625 G6PC2 glucose-6-phosphatase −1.765534746 transferase 1, core 2 catalytic subunit 2 SASS6 SAS-6 centriolar 2.548436625 FUT1 fucosyltransferase 1 −1.765534746 assembly protein (H blood group) AMIGO3 adhesion molecule with 2.548436625 ZFP69 ZFP69 zinc finger protein −1.765534746 Ig-like domain 3 APH1B aph-1 homolog B, 2.548436625 WBSCR27 Williams Beuren syndrome −1.765534746 gamma-secretase subunit chromosome region 27 ABCC5 ATP binding cassette 2.547846505 METTL8 methyltransferase like 8 −1.766880868 subfamily C member 5 YIPF6 Yip1 domain family 2.543805176 TMEM170 transmembrane protein −1.767462508 member 6 170(Tmem170) FFAR1 free fatty acid receptor 1 2.5360529 TRP53INP1 transformation related −1.767518474 protein 53 inducible nuclear protein 1(Trp53inp1) TSSK6 testis specific serine 2.5360529 H2-Q5 histocompatibility 2, Q −1.769676967 kinase 6 region locus 5(H2-Q5) ETV6 ETS variant 6 2.535385323 ADCK1 aarF domain containing −1.770033995 kinase 1 PTGDS prostaglandin D2 2.529838423 IMPAD1 inositol monophosphatase −1.771434505 synthase domain containing 1 SH3D19 SH3 domain containing 2.523561956 E4F1 E4F transcription factor 1 −1.772427885 19 KIF5C kinesin family member 2.518298014 ZFYVE20 Description Not Found −1.772942676 5C PTGER2 prostaglandin E receptor 2 2.517275693 PNPLA6 patatin like phospholipase −1.775074114 domain containing 6 INSR insulin receptor 2.510961919 TRIB3 tribbles pseudokinase 3 −1.775215233 MAPK6 mitogen-activated protein 2.504620392 GM614 predicted gene 614(Gm614) −1.776103988 kinase 6 OXSR1 oxidative stress 2.502211192 D5ERTD579E DNA segment, Chr 5, −1.776306798 responsive 1 ERATO Doi 579, expressed(D5Ertd579e) EZH2 enhancer of zeste 2 2.501439145 SCAND1 SCAN domain containing 1 −1.77785827 polycomb repressive complex 2 subunit BNIP1 BCL2 interacting protein 2.498250868 ASB13 ankyrin repeat and SOCS −1.782205107 1 box containing 13 LPCAT4 lysophosphatidylcholine 2.495285165 ARHGEF4 Rho guanine nucleotide −1.784072601 acyltransferase 4 exchange factor 4 PPAP2C Description Not Found 2.485426827 H1FNT H1 histone family member −1.78485543 N, testis specific IFNA12 interferon alpha 2.485426827 BLOC1S1 biogenesis of lysosomal −1.784911393 12(Ifna12) organelles complex 1 subunit 1 DCLK1 doublecortin like kinase 1 2.485426827 ZFYVE27 zinc finger FYVE-type −1.7851013 containing 27 MX1 MX dynamin like GTPase 2.485426827 RHOX4B reproductive homeobox −1.786596362 1 4B(Rhox4b) SMTN smoothelin 2.485426827 OLFR1134 olfactory receptor −1.786596362 1134(Olfr1134) PLA2G15 phospholipase A2 group 2.48194563 CAR11 carbonic anhydrase −1.786596362 XV 11(Car11) OLFR192 olfactory receptor 2.472487771 LRRIQ4 leucine rich repeats and IQ −1.786596362 192(Olfr192) motif containing 4 ITGB5 integrin subunit beta 5 2.472487771 CASP12 caspase 12 −1.786596362 (gene/pseudogene) RAPSN receptor associated 2.465974465 ODF3L1 outer dense fiber of sperm −1.786596362 protein of the synapse tails 3 like 1 SNX3 sorting nexin 3 2.459431619 CCDC3 coiled-coil domain −1.786596362 containing 3 FERMT2 fermitin family member 2 2.459431619 SSPN sarcospan −1.786596362 CCR5 C-C motif chemokine 2.444410478 KLK1 kallikrein 1 −1.786596362 receptor 5 (gene/pseudogene) UPK1A uroplakin 1A 2.439623138 SENP7 SUMO1/sentrin specific −1.786897131 peptidase 7 BCL2L2 BCL2 like 2 2.43629512 CAML calcium modulating −1.787735284 ligand(Caml) 2610002M06RIK Description Not Found 2.432959407 YEATS2 YEATS domain containing −1.788627083 2 CENPN centromere protein N 2.432959407 SERPINF2 serpin family F member 2 −1.791814071 HBEGF heparin binding EGF like 2.43096254 KCNMB1 potassium calcium- −1.792597191 growth factor activated channel subfamily M regulatory beta subunit 1 TYMS thymidylate synthetase 2.427103287 FCHO2 FCH domain only 2 −1.792666489 MGA MGA, MAX dimerization 2.426939834 BBS9 Bardet-Biedl syndrome 9 −1.792734984 protein RAI14 retinoic acid induced 14 2.426264755 OLFR323 olfactory receptor −1.794609131 323(Olfr323) CFI complement factor I 2.419538892 CD247 CD247 molecule −1.796081585 PLK4 polo like kinase 4 2.419538892 HIST2H2AA1 histone cluster 2, −1.796847743 H2aa1(Hist2h2aa1) SLC6A9 solute carrier family 6 2.419538892 PDK1 pyruvate dehydrogenase −1.800563818 member 9 kinase 1 TMED2 transmembrane p24 2.419538892 NRARP NOTCH-regulated ankyrin −1.803049246 trafficking protein 2 repeat protein TMEM120B transmembrane protein 2.41857423 BTBD11 BTB domain containing 11 −1.804793263 120B TRIM36 tripartite motif containing 2.417852515 CSF2RA colony stimulating factor 2 −1.805089518 36 receptor alpha subunit CCDC93 coiled-coil domain 2.416164165 DEXI Dexi homolog −1.806998156 containing 93 SLC25A35 solute carrier family 25 2.409367225 OLFR1276 olfactory receptor −1.807354922 member 35 1276(Olfr1276) BNC1 basonuclin 1 2.40599236 TCSTV3 2-cell-stage, variable group, −1.807354922 member 3(Tcstv3) FOXL2 forkhead box L2 2.40599236 SPRR2D small proline rich protein −1.807354922 2D TFPI2 tissue factor pathway 2.40599236 SEMA4G semaphorin 4G −1.807354922 inhibitor 2 NET1 neuroepithelial cell 2.40599236 KCNK9 potassium two pore domain −1.807354922 transforming 1 channel subfamily K member 9 SLCO2A1 solute carrier organic 2.40599236 SNAPC3 small nuclear RNA −1.807385513 anion transporter family activating complex member 2A1 polypeptide 3 A730008H23RIK Description Not Found 2.399275037 AXIN2 axin 2 −1.808429403 CDKN2B cyclin dependent kinase 2.397264578 PCNXL3 Description Not Found −1.808995133 inhibitor 2B ZFP532 zinc finger protein 2.393138801 KLHL7 kelch like family member 7 −1.809016035 532(Zfp532) GTSE1 G2 and S-phase expressed 2.392428431 ZFP281 zinc finger protein −1.811556991 1 281(Zfp281) CCDC14 coiled-coil domain 2.392317423 CHRNB2 cholinergic receptor −1.812498225 containing 14 nicotinic beta 2 subunit ADAT1 adenosine deaminase, 2.392317423 TBC1D15 TBC1 domain family −1.812909044 tRNA specific 1 member 15 DGKH diacylglycerol kinase eta 2.392317423 GALNT9 polypeptide N- −1.813407449 acetylgalactosaminyl- transferase 9 ZRSR1 zinc finger CCCH-type, 2.392317423 DYNC1I1 dynein cytoplasmic 1 −1.813434179 RNA binding motif and intermediate chain 1 serine/arginine rich 1 NFE2 nuclear factor, erythroid 2 2.391529377 MYH8 myosin heavy chain 8 −1.81403224 CD63 CD63 molecule 2.387853137 CEP57 centrosomal protein 57 −1.815684972 MIB1 mindbomb E3 ubiquitin 2.38645559 LTK leukocyte receptor tyrosine −1.817623258 protein ligase 1 kinase TSN translin 2.382349023 COMMD2 COMM domain containing −1.817623258 2 2510003E04RIK Description Not Found 2.378511623 MEF2C myocyte enhancer factor 2C −1.817623258 BC043934 cDNA sequence 2.378511623 LONRF2 LON peptidase N-terminal −1.817941412 BC043934(BC043934) domain and ring finger 2 AHCYL1 adenosylhomocysteinase 2.366734247 PDCD6IP programmed cell death 6 −1.820575529 like 1 interacting protein OLFR731 olfactory receptor 2.364572432 DHX16 DEAH-box helicase 16 −1.820661084 731(Olfr731) CDKN2A cyclin dependent kinase 2.364572432 ZFYVE19 zinc finger FYVE-type −1.825281028 inhibitor 2A containing 19 SLC29A4 solute carrier family 29 2.364572432 H2-T10 histocompatibility 2, T −1.826218639 member 4 region locus 10(H2-T10) SLC4A10 solute carrier family 4 2.364572432 ARID1A AT-rich interaction domain −1.827043205 member 10 1A CYCS cytochrome c, somatic 2.351872866 NOD1 nucleotide binding −1.827185706 oligomerization domain containing 1 COL5A1 collagen type V alpha 1 2.350497247 2610318N02RIK Description Not Found −1.827819025 UTRN utrophin 2.350497247 BC048644 cDNA sequence −1.827819025 BC048644(BC048644) AURKA aurora kinase A 2.349678136 CDC42EP2 CDC42 effector protein 2 −1.827819025 KREMEN2 kringle containing 2.349431709 CCL25 C-C motif chemokine −1.827819025 transmembrane protein 2 ligand 25 FGL2 fibrinogen like 2 2.346409407 TBX6 T-box 6 −1.827819025 NCAM1 neural cell adhesion 2.343407822 PLEKHG4 pleckstrin homology and −1.827819025 molecule 1 RhoGEF domain containing G4 ALG8 ALG8, alpha-1,3- 2.343407822 RAD18 RAD18, E3 ubiquitin −1.830642494 glucosyltransferase protein ligase OLFR703 olfactory receptor 2.336283388 SLC12A9 solute carrier family 12 −1.830807586 703(Olfr703) member 9 SLC39A10 solute carrier family 39 2.336283388 NR1D2 nuclear receptor subfamily −1.837943242 member 10 1 group D member 2 HIST1H2AH histone cluster 1, H2ah 2.322141712 NLK nemo like kinase −1.840170811 TSGA8 testis specific gene 2.321928095 TTC37 tetratricopeptide repeat −1.840462743 A8(Tsga8) domain 37 ELOVL2 ELOVL fatty acid 2.321928095 DLG3 discs large MAGUK −1.841507525 elongase 2 scaffold protein 3 MLF1 myeloid leukemia factor 1 2.321928095 PCF11 PCF11 cleavage and −1.843349827 polyadenylation factor subunit FZD6 frizzled class receptor 6 2.321928095 HIST1H4D histone cluster 1, H4d −1.846386944 PLD1 phospholipase D1 2.321928095 PEX26 peroxisomal biogenesis −1.847440096 factor 26 IFRD2 interferon-related 2.321928095 CYP2B10 cytochrome P450, family 2, −1.847996907 developmental regulator 2 subfamily b, polypeptide 10(Cyp2b10) OLA1 Obg-like ATPase 1 2.321928095 GDF3 growth differentiation −1.847996907 factor 3 ASPA aspartoacylase 2.321928095 GPR33 G protein-coupled receptor −1.847996907 33 (gene/pseudogene) TGFB3 transforming growth 2.321928095 TDG thymine DNA glycosylase −1.847996907 factor beta 3 PKIG protein kinase (cAMP- 2.314696526 HIPK3 homeodomain interacting −1.847996907 dependent, catalytic) protein kinase 3 inhibitor gamma TNFRSF4 tumor necrosis factor 2.308832886 PAPOLA poly(A) polymerase alpha −1.847996907 receptor superfamily member 4 IQCB1 IQ motif containing B1 2.307984443 MAPK4 mitogen-activated protein −1.847996907 kinase 4 SLC16A11 solute carrier family 16 2.307662797 FRAT2 frequently rearranged in −1.84969115 member 11 advanced T-cell lymphomas 2 1190002N15RIK Description Not Found 2.307428525 HEXIM1 hexamethylene −1.851035845 bisacetamide inducible 1 LCE1L late cornified envelope 2.307428525 TATDN2 TatD DNase domain −1.851433223 1L(Lce1l) containing 2 RGS13 regulator of G-protein 2.307428525 KLRB1C killer cell lectin-like −1.854253843 signaling 13 receptor subfamily B member 1C(Klrb1c) FBXW8 F-box and WD repeat 2.299987517 SLC16A9 solute carrier family 16 −1.855083462 domain containing 8 member 9 SNCA synuclein alpha 2.296457407 ACBD4 acyl-CoA binding domain −1.855739032 containing 4 OSGIN1 oxidative stress induced 2.294491702 REXO1 RNA exonuclease 1 −1.857980995 growth inhibitor 1 homolog BC004004 cDNA sequence 2.292781749 OLFR1442 olfactory receptor −1.859286959 BC004004(BC004004) 1442(Olfr1442) WNT10A Wnt family member 10A 2.292781749 PHOSPHO1 phosphoethanolamine/ −1.859747926 phosphocholine phosphatase THG1L tRNA-histidine 2.292781749 ITPKA inositol-trisphosphate 3- −1.859881803 guanylyltransferase 1 like kinase A MLH1 mutL homolog 1 2.292781749 ZFHX2 zinc finger homeobox 2 −1.860513882 RRM2 ribonucleotide reductase 2.289435485 TOR1A torsin family 1 member A −1.860949348 regulatory subunit M2 SHISA4 shisa family member 4 2.277984747 CDKAL1 CDK5 regulatory subunit −1.862794137 associated protein 1 like 1 DDAH2 dimethylarginine 2.277984747 SMAD1 SMAD family member 1 −1.863462947 dimethylaminohydrolase 2 APBA1 amyloid beta precursor 2.269085766 ZC3H13 zinc finger CCCH-type −1.863535399 protein binding family A containing 13 member 1 MMAB methylmalonic aciduria 2.264911693 ZSCAN20 zinc finger and SCAN −1.863962106 (cobalamin deficiency) domain containing 20 cblB type DIAP1 Description Not Found 2.263034406 EPB4.1L4A Description Not Found −1.867896464 CAR14 carbonic anhydrase 2.263034406 ZFP280C zinc finger protein −1.867896464 14(Car14) 280C(Zfp280c) C2 complement component 2 2.263034406 GM1322 predicted gene −1.867896464 1322(Gm1322) MAG myelin associated 2.263034406 OLFR472 olfactory receptor −1.867896464 glycoprotein 472(Olfr472) KCNIP3 potassium voltage-gated 2.263034406 OLFR171 olfactory receptor −1.867896464 channel interacting 171(Olfr171) protein 3 CFD complement factor D 2.263034406 OLFR1249 olfactory receptor −1.867896464 1249(Olfr1249) CCNE1 cyclin E1 2.262723645 PRH1 proline rich protein HaeIII −1.867896464 subfamily 1 RYR1 ryanodine receptor 1 2.261305322 ARSI arylsulfatase family −1.867896464 member I PROC protein C, inactivator of 2.255500733 KRT7 keratin 7 −1.867896464 coagulation factors Va and VIIIa ZFP27 zinc finger protein 2.247927513 PCGF3 polycomb group ring finger −1.867896464 27(Zfp27) 3 TBX1 T-box 1 2.247927513 PCTP phosphatidylcholine −1.867896464 transfer protein DHRS13 dehydrogenase/reductase 2.247927513 CALD1 caldesmon 1 −1.867896464 13 HSPG2 heparan sulfate 2.247927513 TREML2 triggering receptor −1.867896464 proteoglycan 2 expressed on myeloid cells like 2 FRMD8 FERM domain containing 2.24777312 RTN4RL1 reticulon 4 receptor like 1 −1.867896464 8 MIOX myo-inositol oxygenase 2.240579987 PARVA parvin alpha −1.868479018 LYRM1 LYR motif containing 1 2.232660757 NPCD neuronal pentraxin chromo −1.871902039 domain(Npcd) STAP1 signal transducing adaptor 2.232660757 RFXANK regulatory factor X −1.87206109 family member 1 associated ankyrin containing protein NAT2 N-acetyltransferase 2 2.232660757 MAP3K14 mitogen-activated protein −1.872291304 kinase kinase kinase 14 SRGAP3 SLIT-ROBO Rho GTPase 2.232660757 KLHL9 kelch like family member 9 −1.874528943 activating protein 3 NXT2 nuclear transport factor 2 2.232660757 SESN1 sestrin 1 −1.875260951 like export factor 2 RCOR1 REST corepressor 1 2.232660757 ADAMTS7 ADAM metallopeptidase −1.879404807 with thrombospondin type 1 motif 7 SRR serine racemase 2.230836503 SNAPC1 small nuclear RNA −1.88488993 activating complex polypeptide 1 IKBKAP inhibitor of kappa light 2.226177109 ADAR adenosine deaminase, RNA −1.885299379 polypeptide gene specific enhancer in B-cells, kinase complex- associated protein AI597479 expressed sequence 2.225819675 LCE1C late cornified envelope 1C −1.885626461 AI597479(AI597479) POP1 POP1 homolog, 2.224966365 FBXO21 F-box protein 21 −1.886155099 ribonuclease P/MRP subunit SLC35E4 solute carrier family 35 2.217230716 2610524H06RIK Description Not Found −1.887525271 member E4 XAB2 XPA binding protein 2 2.217230716 1700016K19RIK Description Not Found −1.887525271 MREG melanoregulin 2.2129258 ZFP715 zinc finger protein −1.887525271 715(Zfp715) FKBP11 FK506 binding protein 11 2.210721954 OLFR446 olfactory receptor −1.887525271 446(Olfr446) IGF2BP2 insulin like growth factor 2.207789851 PTK7 protein tyrosine kinase 7 −1.887525271 2 mRNA binding protein (inactive) 2 NUP133 nucleoporin 133 2.207447199 TMEM117 transmembrane protein 117 −1.887525271 OLFR1183 olfactory receptor 2.201633861 ITIH2 inter-alpha-trypsin inhibitor −1.887525271 1183(Olfr1183) heavy chain 2 IL1F6 interleukin 1 family, 2.201633861 TAGLN3 transgelin 3 −1.887525271 member 6(II1f6) OTX1 orthodenticle homeobox 1 2.201633861 IFI203 interferon activated gene −1.887644112 203(Ifi203) MSH3 mutS homolog 3 2.201633861 ATP1B1 ATPase Na+/K+ −1.887664186 transporting subunit beta 1 SCN4B sodium voltage-gated 2.201633861 BLCAP bladder cancer associated −1.888596201 channel beta subunit 4 protein CROCC ciliary rootlet coiled-coil, 2.201633861 IGF1R insulin like growth factor 1 −1.89024137 rootletin receptor NSUN2 NOP2/Sun RNA 2.194349986 HMG20A high mobility group 20A −1.890579593 methyltransferase family member 2 GAS2L1 growth arrest specific 2 2.193771743 WDR24 WD repeat domain 24 −1.891527175 like 1 3110007F17RIK Description Not Found 2.190740399 CDX4 caudal type homeobox 4 −1.892655439 DEFB15 defensin beta 15(Defb15) 2.185866545 CLDN18 claudin 18 −1.893449375 C1QTNF2 C1q and tumor necrosis 2.185866545 IL4RA interleukin 4 receptor, −1.895369594 factor related protein 2 alpha(Il4ra) RAP1GAP RAP1 GTPase activating 2.185866545 RETNLA resistin like alpha(Retnla) −1.895739477 protein SNTB1 syntrophin beta 1 2.185866545 AA388235 expressed sequence −1.895739477 AA388235(AA388235) FAH fumarylacetoacetate 2.182925576 ZC3H6 zinc finger CCCH-type −1.896127489 hydrolase containing 6 AVPI1 arginine vasopressin 2.174393775 D930015E06RIK RIKEN cDNA −1.899656973 induced 1 D930015E06 gene(D930015E06Rik) RPA2 replication protein A2 2.172751912 NPFFR2 neuropeptide FF receptor 2 −1.902073579 BRCA2 BRCA2, DNA repair 2.168732488 IRAK1 interleukin 1 receptor −1.90243374 associated associated kinase 1 RBM47 RNA binding motif 2.165911939 CWF19L2 CWF19-like 2, cell cycle −1.903704505 protein 47 control (S. pombe) MSL3L2 male-specific lethal 3-like 2.159061455 STK40 serine/threonine kinase 40 −1.903964448 2 (Drosophila)(Ms1312) TNFRSF9 tumor necrosis factor 2.156071704 MARS2 methionyl-tRNA synthetase −1.904571951 receptor superfamily 2, mitochondrial member 9 TRF transferrin(Trf) 2.154588207 RAB5A RAB5A, member RAS −1.906350687 oncogene family ZDHHC15 zinc finger DHHC-type 2.154372546 OLFR1037 olfactory receptor −1.906890596 containing 15 1037(Olfr1037) IGJ Description Not Found 2.153805336 ARHGAP22 Rho GTPase activating −1.906890596 protein 22 FBXO27 F-box protein 27 2.153805336 DENND1B DENN domain containing −1.906890596 1B ZDHHC24 zinc finger DHHC-type 2.153805336 EAPP E2F associated −1.906890596 containing 24 phosphoprotein SPCS2 signal peptidase complex 2.153805336 ANKRD13D ankyrin repeat domain 13D −1.906890596 subunit 2 UCN3 urocortin 3 2.153805336 EFCAB2 EF-hand calcium binding −1.906890596 domain 2 SLC35A1 solute carrier family 35 2.153805336 HOXC9 homeobox C9 −1.906890596 member A1 PODXL podocalyxin like 2.153805336 SENP6 SUMO1/sentrin specific −1.907956932 peptidase 6 FAM154B Description Not Found 2.153792145 SIDT1 SID1 transmembrane −1.908286674 family member 1 NRP1 neuropilin 1 2.147470553 2310057J18RIK Description Not Found −1.916476644 ERGIC1 endoplasmic reticulum- 2.147104727 SPRYD4 SPRY domain containing 4 −1.916476644 golgi intermediate compartment 1 RNF26 ring finger protein 26 2.146810011 LY6D lymphocyte antigen 6 −1.916476644 complex, locus D LCN3 lipocalin 3(Lcn3) 2.137503524 PPARGC1B PPARG coactivator 1 beta −1.917291956 FMO1 flavin containing 2.137503524 SH3TC1 SH3 domain and −1.917906346 monooxygenase 1 tetratricopeptide repeats 1 RAB20 RAB20, member RAS 2.137503524 FOXO1 forkhead box O1 −1.920209106 oncogene family KATNAL1 katanin catalytic subunit 2.137503524 DHX40 DEAH-box helicase 40 −1.920623917 A1 like 1 GPR107 G protein-coupled 2.136424717 RECQL5 RecQ like helicase 5 −1.920664575 receptor 107 MELK maternal embryonic 2.133399125 RBM15 RNA binding motif protein −1.922616041 leucine zipper kinase 15 KCTD9 potassium channel 2.13207329 EGLN2 egl-9 family hypoxia −1.924079933 tetramerization domain inducible factor 2 containing 9 PBK PDZ binding kinase 2.130417144 GPR112 Description Not Found −1.925999419 ENPP5 ectonucleotide 2.124112676 OLFR829 olfactory receptor −1.925999419 pyrophosphatase/ 829(Olfr829) phosphodiesterase 5 (putative) ZDHHC16 zinc finger DHHC-type 2.12361008 OLFR684 olfactory receptor −1.925999419 containing 16 684(Olfr684) OLFR1346 olfactory receptor 2.121015401 RETN resistin −1.925999419 1346(Olfr1346) MILL1 MHC I like leukocyte 2.121015401 ST6GALNAC2 ST6 N- −1.925999419 1(Mill1) acetylgalactosaminide alpha-2,6-sialyltransferase 2 RHCG Rh family C glycoprotein 2.121015401 FES FES proto-oncogene, −1.925999419 tyrosine kinase CLDN1 claudin 1 2.121015401 KIF13A kinesin family member 13A −1.925999419 LHX3 LIM homeobox 3 2.121015401 TRPT1 tRNA phosphotransferase 1 −1.926457816 TUBB2A tubulin beta 2A class IIa 2.121015401 PLCB2 phospholipase C beta 2 −1.927343833 GSG2 germ cell associated 2, 2.119412265 NADSYN1 NAD synthetase 1 −1.929674394 haspin HYAL2 hyaluronoglucosaminidase 2.107345942 4833420G17RIK Description Not Found −1.93060469 2 1700003F12RIK Description Not Found 2.10433666 P2RY10 purinergic receptor P2Y10 −1.930737338 RUSC2 RUN and SH3 domain 2.10433666 PPAPDC3 Description Not Found −1.935459748 containing 2 LRRIQ3 leucine rich repeats and 2.10433666 DIP2B disco interacting protein 2 −1.935459748 IQ motif containing 3 homolog B CHSY1 chondroitin sulfate 2.10433666 RHAG Rh-associated glycoprotein −1.935459748 synthase 1 DUSP23 dual specificity 2.10433666 EMID1 EMI domain containing 1 −1.935459748 phosphatase 23 RRAGB Ras related GTP binding 2.10433666 RNF4 ring finger protein 4 −1.938834579 B KCNAB3 potassium voltage-gated 2.10433666 UBL5 ubiquitin like 5 −1.938952478 channel subfamily A regulatory beta subunit 3 GRPEL2 GrpE like 2, 2.103129681 PROSC proline synthetase −1.94016675 mitochondrial cotranscribed homolog (bacterial) TRAF2 TNF receptor associated 2.102029095 FZD5 frizzled class receptor 5 −1.942503137 factor 2 COQ7 coenzyme Q7, 2.100205246 UBE2D1 ubiquitin conjugating −1.942775467 hydroxylase enzyme E2 D1 TMEM126B transmembrane protein 2.099187297 KLRA7 killer cell lectin-like −1.943510757 126B receptor, subfamily A, member 7(Klra7) SGPL1 sphingosine-1-phosphate 2.097112667 TMEM63C transmembrane protein 63C −1.94425562 lyase 1 CAPN2 calpain 2 2.096447979 2810006K23RIK Description Not Found −1.944858446 CHEK2 checkpoint kinase 2 2.088457439 OLFR672 olfactory receptor −1.944858446 672(Olfr672) GLRP1 glutamine repeat protein 2.087462841 OLFR1347 olfactory receptor −1.944858446 1(Glrp1) 1347(Olfr1347) RTN4R reticulon 4 receptor 2.087462841 MTTP microsomal triglyceride −1.944858446 transfer protein TRIM37 tripartite motif containing 2.087462841 MSX1 msh homeobox 1 −1.944858446 37 NUCB2 nucleobindin 2 2.087462841 BSND barttin CLCNK type −1.944858446 accessory beta subunit UBE2T ubiquitin conjugating 2.073616696 MARK1 microtubule affinity −1.944858446 enzyme E2 T regulating kinase 1 CREB3L3 CAMP responsive element 2.070389328 CHRNB1 cholinergic receptor −1.944858446 binding protein 3 like 3 nicotinic beta 1 subunit CHRM4 cholinergic receptor 2.070389328 CRYL1 crystallin lambda 1 −1.946419425 muscarinic 4 SLC16A13 solute carrier family 16 2.070389328 TEC tec protein tyrosine kinase −1.947330641 member 13 OLFML2B olfactomedin like 2B 2.070389328 XKR6 XK related 6 −1.95031589 CSNK1G1 casein kinase 1 gamma 1 2.070389328 ARC activity-regulated −1.953636949 cytoskeleton-associated protein S100A14 S100 calcium binding 2.070389328 WFDC10 WAP four-disulfide core −1.95419631 protein A14 domain 10(Wfdc10) SMYD4 SET and MYND domain 2.070389328 OLFR866 olfactory receptor −1.959768144 containing 4 866(Olfr866) CH25H cholesterol 25- 2.070389328 WIPI2 WD repeat domain, −1.960171668 hydroxylase phosphoinositide interacting 2 TEX2 testis expressed 2 2.067875748 OLFR948 olfactory receptor −1.963474124 948(Olfr948) SYN1 synapsin I 2.063429187 CRTAM cytotoxic and regulatory T- −1.963474124 cell molecule CYP3A13 cytochrome P450, family 2.060581758 CCDC116 coiled-coil domain −1.963474124 3, subfamily a, containing 116 polypeptide 13(Cyp3a13) CBX8 chromobox 8 2.060297534 ALAS2 5′-aminolevulinate synthase −1.963474124 2 TOR2A torsin family 2 member A 2.056535553 SDC4 syndecan 4 −1.963474124 E230025N22RIK Riken cDNA 2.053111336 LENG1 leukocyte receptor cluster −1.963474124 E230025N22 member 1 gene(E230025N22Rik) OLFR963 olfactory receptor 2.053111336 TRIM65 tripartite motif containing −1.963474124 963(Olfr963) 65 OLFR694 olfactory receptor 2.053111336 ADRA2B adrenoceptor alpha 2B −1.963474124 694(Olfr694) AKR1B8 aldo-keto reductase 2.053111336 CPSF4 cleavage and −1.964016356 family 1, member polyadenylation specific B8(Akr1b8) factor 4 UGDH UDP-glucose 6- 2.053111336 LRCH1 leucine rich repeats and −1.966068313 dehydrogenase calponin homology domain containing 1 CLPB ClpB homolog, 2.053111336 CPXM1 carboxypeptidase X (M14 −1.96782195 mitochondrial AAA family), member 1 ATPase chaperonin KLHDC9 kelch domain containing 2.053111336 PARP6 poly(ADP-ribose) −1.968362498 9 polymerase family member 6 MCPH1 microcephalin 1 2.051211057 GTF3C2 general transcription factor −1.975687807 IIIC subunit 2 IL2RA interleukin 2 receptor 2.049225103 NEDD4L neural precursor cell −1.978518523 subunit alpha expressed, developmentally down-regulated 4-like, E3 ubiquitin protein ligase CAR9 carbonic anhydrase 2.044394119 DICER1 dicer 1, ribonuclease III −1.97959126 9(Car9) USP10 ubiquitin specific 2.044394119 GBA2 glucosylceramidase beta 2 −1.980387638 peptidase 10 FASTKD2 FAST kinase domains 2 2.044394119 OLFR1269 olfactory receptor −1.981852653 1269(Olfr1269) STRA13 stimulated by retinoic 2.044394119 EAR10 eosinophil-associated, −1.981852653 acid 13 ribonuclease A family, member 10(Ear10) HIST1H2AD histone cluster 1, H2ad 2.044111161 ADAM5 ADAM metallopeptidase −1.981852653 domain 5 (pseudogene) PLA1A phospholipase A1 2.037157781 MED1 mediator complex subunit 1 −1.981852653 member A MCM3 minichromosome 2.036462274 FGFRL1 fibroblast growth factor −1.981852653 maintenance complex receptor-like 1 component 3 PIF1 PIF1 5′-to-3′ DNA 2.036094966 EXTL1 exostosin like −1.981852653 helicase glycosyltransferase 1 GALR1 galanin receptor 1 2.03562391 ZFHX3 zinc finger homeobox 3 −1.981852653 DLD dihydrolipoamide 2.03562391 FBXO30 F-box protein 30 −1.981852653 dehydrogenase GGCX gamma-glutamyl 2.03562391 RNF112 ring finger protein 112 −1.984681148 carboxylase CEP68 centrosomal protein 68 2.03562391 PARP3 poly(ADP-ribose) −1.98599548 polymerase family member 3 MMP11 matrix metallopeptidase 2.03562391 AIRE autoimmune regulator −1.986410935 11 STMN1 stathmin 1 2.033316653 CYB561D1 cytochrome b561 family −1.987107951 member D1 SLCO4A1 solute carrier organic 2.03217627 TRAPPC5 trafficking protein particle −1.987269174 anion transporter family complex 5 member 4A1 TIAL1 TIA1 cytotoxic granule- 2.02888965 RFTN2 raftlin family member 2 −1.98749308 associated RNA binding protein-like 1 0610009B22RIK Description Not Found 2.017921908 FRAT1 frequently rearranged in −1.999894159 advanced T-cell lymphomas 1 GM1673 predicted gene 2.017921908 DMC1 DNA meiotic recombinase −2 1673(Gm1673) 1 CCL26 C-C motif chemokine 2.017921908 RIPK4 receptor interacting −2 ligand 26 serine/threonine kinase 4 ZWILCH zwilch kinetochore 2.017921908 PVR poliovirus receptor −2 protein GABRA1 gamma-aminobutyric acid 2.017921908 LPIN2 lipin 2 −2 type A receptor alpha1 subunit ACP2 acid phosphatase 2, 2.017143376 THAP2 THAP domain containing 2 −2 lysosomal FAM131A family with sequence 2.013219985 SHE Src homology 2 domain −2 similarity 131 member A containing E PXMP4 peroxisomal membrane 2.012497517 ARHGAP25 Rho GTPase activating −2.005618551 protein 4 protein 25 CDC6 cell division cycle 6 2.011166077 CSF1R colony stimulating factor 1 −2.006350699 receptor AXL AXL receptor tyrosine 2.008131619 ZFP1 ZFP1 zinc finger protein −2.007904843 kinase RBBP7 RB binding protein 7, 2.006746832 SFN stratifin −2.008988783 chromatin remodeling factor PABPC4 poly(A) binding protein 2.005260152 COL17A1 collagen type XVII alpha 1 −2.010386372 cytoplasmic 4 HIST1H2AK histone cluster 1, H2ak 2.003307679 XKRX XK related, X-linked −2.0105696 MTFMT mitochondrial methionyl- 2.001754595 BRD8 bromodomain containing 8 −2.01346226 tRNA formyltransferase ZFP449 zinc finger protein 2 ZFP213 zinc finger protein −2.013532276 449(Zfp449) 213(Zfp213) D930020B18RIK RIKEN cDNA 2 ZFY2 zinc finger protein 2, Y- −2.015657249 D930020B18 linked(Zfy2) gene(D930020B18Rik) LCE1D late cornified envelope 2 MAP3K3 mitogen-activated protein −2.01612652 1D kinase kinase kinase 3 UCN urocortin 2 ZFP445 zinc finger protein −2.017921908 445(Zfp445) SYT4 synaptotagmin 4 2 MTAP7D3 MAP7 domain containing −2.017921908 3(Mtap7d3) GPR132 G protein-coupled 2 TMPRSS11A transmembrane protease, −2.017921908 receptor 132 serine 11A SDHD succinate dehydrogenase 2 OLFM2 olfactomedin 2 −2.017921908 complex subunit D PANK3 pantothenate kinase 3 2 GRM4 glutamate metabotropic −2.017921908 receptor 4 SBSN suprabasin 1.99095486 ONECUT2 one cut homeobox 2 −2.017921908 WDR59 WD repeat domain 59 1.989976974 HNRNPH3 heterogeneous nuclear −2.017921908 ribonucleoprotein H3 MTMR9 myotubularin related 1.987844644 ZMYM5 zinc finger MYM-type −2.020204421 protein 9 containing 5 IL15RA interleukin 15 receptor 1.985628881 RAPGEF6 Rap guanine nucleotide −2.020953989 subunit alpha exchange factor 6 RHBDF2 rhomboid 5 homolog 2 1.984681148 CD34 CD34 molecule −2.026714044 NHLRC2 NHL repeat containing 2 1.98375117 ACVR2B activin A receptor type 2B −2.026714044 NMRAL1 NmrA-like family domain 1.983370163 RILP Rab interacting lysosomal −2.026800059 containing 1 protein OLFR120 olfactory receptor 1.981852653 EMR1 Description Not Found −2.031218731 120(Olfr120) OLFR1051 olfactory receptor 1.981852653 DNAJA2 DnaJ heat shock protein −2.031291874 1051(Olfr1051) family (Hsp40) member A2 PCDHGA9 protocadherin gamma 1.981852653 SEMA4B semaphorin 4B −2.031985281 subfamily A, 9 FST follistatin 1.981852653 1700015E13RIK Description Not Found −2.03562391 RECQL4 RecQ like helicase 4 1.976611605 RHOX1 reproductive homeobox −2.03562391 1(Rhox1) NFKBIL1 NFKB inhibitor like 1 1.970969489 TCP11 t-complex 11 −2.03562391 TUBD1 tubulin delta 1 1.964367355 FBXW11 F-box and WD repeat −2.03562391 domain containing 11 FSD1 fibronectin type III and 1.963474124 ALX1 ALX homeobox 1 −2.03562391 SPRY domain containing 1 GDF5 growth differentiation 1.963474124 BST1 bone marrow stromal cell −2.03562391 factor 5 antigen 1 TREML4 triggering receptor 1.963474124 GPR83 G protein-coupled receptor −2.03562391 expressed on myeloid 83 cells like 4 SORD sorbitol dehydrogenase 1.963474124 RECK reversion inducing cysteine −2.036112118 rich protein with kazal motifs HEBP1 heme binding protein 1 1.963474124 ABHD14B abhydrolase domain −2.040460993 containing 14B KDELR2 KDEL endoplasmic 1.96155465 GPRC6A G protein-coupled receptor −2.042122888 reticulum protein class C group 6 member A retention receptor 2 TRPV4 transient receptor 1.958842675 GRAMD3 GRAM domain containing −2.042296131 potential cation channel 3 subfamily V member 4 ABHD5 abhydrolase domain 1.957389419 IMPACT impact RWD domain −2.042436285 containing 5 protein YOD1 YOD1 deubiquitinase 1.95419631 TOP1 topoisomerase (DNA) I −2.044394119 MAGOHB mago homolog B, exon 1.952932368 NACC2 NACC family member 2 −2.044394119 junction complex core component TSPAN2 tetraspanin 2 1.95176103 PKNOX1 PBX/knotted 1 homeobox 1 −2.045797958 LDB3 LIM domain binding 3 1.94850842 TMEM79 transmembrane protein 79 −2.046628729 1700067P10RIK Description Not Found 1.944858446 MYCBP2 MYC binding protein 2, E3 −2.047368853 ubiquitin protein ligase 9530091C08RIK Description Not Found 1.944858446 MAS1 MAS1 proto-oncogene, G −2.048055651 protein-coupled receptor RHOJ ras homolog family 1.944858446 GEMIN6 gem nuclear organelle −2.053111336 member J associated protein 6 SFRP1 secreted frizzled related 1.944858446 TMEM100 transmembrane protein 100 −2.053111336 protein 1 XPNPEP2 X-prolyl aminopeptidase 1.944858446 FOXI1 forkhead box I1 −2.053111336 (aminopeptidase P) 2, membrane-bound RNASE4 ribonuclease A family 1.935459748 OPLAH 5-oxoprolinase (ATP- −2.053111336 member 4 hydrolysing) NAPSA napsin A aspartic 1.931586931 BC094916 Description Not Found −2.058337935 peptidase TIMM22 translocase of inner 1.931202999 GZMM granzyme M −2.061193332 mitochondrial membrane 22 homolog (yeast) MTCH2 mitochondrial carrier 2 1.929774464 RCOR2 REST corepressor 2 −2.06280495 ADCK4 aarF domain containing 1.927921426 NR2E1 nuclear receptor subfamily −2.06366268 kinase 4 2 group E member 1 PDSS1 prenyl (decaprenyl) 1.926245513 NT5DC1 5′-nucleotidase domain −2.065994119 diphosphate synthase, containing 1 subunit 1 ZFP94 zinc finger protein 1.925999419 SCN8A sodium voltage-gated −2.06750099 94(Zfp94) channel alpha subunit 8 FABP9 fatty acid binding protein 1.925999419 CBX7 chromobox 7 −2.06750099 9 RNF170 ring finger protein 170 1.925999419 FHAD1 forkhead associated −2.068114527 phosphopeptide binding domain 1 TLR3 toll like receptor 3 1.925999419 KCNQ3 potassium voltage-gated −2.068885643 channel subfamily Q member 3 LIPH lipase H 1.925999419 BC025920 zinc finger protein −2.070389328 pseudogene(BC025920) PLEKHA7 pleckstrin homology 1.925999419 FCGR1 Fc receptor, IgG, high −2.070389328 domain containing A7 affinity I(Fcgr1) LXN latexin 1.9244606 SYN3 synapsin III −2.070389328 PPCS phosphopantothenoylcyst 1.92294738 KLHL5 kelch like family member 5 −2.070389328 eine synthetase BTRC beta-transducin repeat 1.92065845 EDA2R ectodysplasin A2 receptor −2.070389328 containing E3 ubiquitin protein ligase APIP APAF1 interacting 1.920326443 STK38 serine/threonine kinase 38 −2.070389328 protein ANK1 ankyrin 1 1.916476644 CDKN2D cyclin dependent kinase −2.072205467 inhibitor 2D TOMM70A translocase of outer 1.913107017 IL6ST interleukin 6 signal −2.072660321 mitochondrial membrane transducer 70 homolog A (yeast)(Tomm70a) ABCB1B ATP-binding cassette, 1.908033945 OLFR427 olfactory receptor −2.074318985 sub-family B 427(Olfr427) (MDR/TAP), member 1B(Abcb1b) ACN9 Description Not Found 1.906890596 BAIAP2 BAIl associated protein 2 −2.078951341 DLX1AS distal-less homeobox 1, 1.906890596 TIMP2 TIMP metallopeptidase −2.079805224 antisense(Dlx1as) inhibitor 2 MRGPRD MAS related GPR family 1.906890596 CDCP1 CUB domain containing −2.083991945 member D protein 1 WDHD1 WD repeat and HMG-box 1.906890596 RGS14 regulator of G-protein −2.084198537 DNA binding protein 1 signaling 14 USP46 ubiquitin specific 1.906890596 VASP vasodilator-stimulated −2.086359868 peptidase 46 phosphoprotein PKN3 protein kinase N3 1.906890596 ZFP318 zinc finger protein −2.087462841 318(Zfp318) OSCAR osteoclast associated, 1.906890596 PSG25 pregnancy-specific −2.087462841 immunoglobulin-like glycoprotein 25(Psg25) receptor CDK2 cyclin dependent kinase 2 1.906746727 PDZD8 PDZ domain containing 8 −2.087462841 TRIM62 tripartite motif containing 1.905520967 DET1 de-etiolated homolog 1 −2.087462841 62 (Arabidopsis) SQLE squalene epoxidase 1.903767694 CHST3 carbohydrate −2.087462841 sulfotransferase 3 MCM10 minichromosome 1.89598378 EHHADH enoyl-CoA, hydratase/3- −2.087462841 maintenance 10 hydroxyacyl CoA replication initiation dehydrogenase factor CCDC90B coiled-coil domain 1.894803124 FCGRT Fc fragment of IgG receptor −2.090735607 containing 90B and transporter SPATS1 spermatogenesis 1.892848083 CFP complement factor −2.09437407 associated serine rich 1 properdin GPNMB glycoprotein nmb 1.891427809 SOCS6 suppressor of cytokine −2.094638136 signaling 6 MST1 macrophage stimulating 1 1.88993148 SYT11 synaptotagmin 11 −2.09592442 LTB4R1 leukotriene B4 receptor 1.887644112 MBTPS2 membrane bound −2.09592442 1(Ltb4r1) transcription factor peptidase, site 2 DNAJC5B DnaJ heat shock protein 1.887525271 MEFV Mediterranean fever −2.097059135 family (Hsp40) member C5 beta PCDHGC4 protocadherin gamma 1.887525271 SRPK2 SRSF protein kinase 2 −2.10044313 subfamily C, 4 HMX2 H6 family homeobox 2 1.887525271 DUSP16 dual specificity phosphatase −2.102740277 16 NDUFAB1 NADH:ubiquinone 1.887525271 SLC6A7 solute carrier family 6 −2.103129681 oxidoreductase subunit member 7 AB1 MGP matrix Gla protein 1.887525271 HBB-B1 hemoglobin, beta adult −2.10433666 major chain(Hbb-b1) ZKSCAN2 zinc finger with KRAB 1.887525271 TNPO3 transportin 3 −2.10433666 and SCAN domains 2 CCDC51 coiled-coil domain 1.887525271 CSNK2B casein kinase 2 beta −2.10433666 containing 51 CTSK cathepsin K 1.887525271 BCAS1 breast carcinoma amplified −2.10433666 sequence 1 PRDM9 PR domain 9 1.887525271 INO80 INO80 complex subunit −2.10433666 C8A complement component 8 1.887525271 MPG N-methylpurine DNA −2.10433666 alpha subunit glycosylase NEUROG1 neurogenin 1 1.887082413 FOXP1 forkhead box P1 −2.107557734 NUSAP1 nucleolar and spindle 1.886951242 USP21 ubiquitin specific peptidase −2.107658353 associated protein 1 21 LZIC leucine zipper and 1.877899051 LIMS1 LIM zinc finger domain −2.112700133 CTNNBIP1 domain containing 1 containing ZFP609 zinc finger protein 1.87774425 FXYD1 FXYD domain containing −2.112700133 609(Zfp609) ion transport regulator 1 GPR87 G protein-coupled 1.87774425 POU3F1 POU class 3 homeobox 1 −2.113574207 receptor 87 GMPPB GDP-mannose 1.871523637 OLFR591 olfactory receptor −2.114494844 pyrophosphorylase B 591(Olfr591) TMEM115 transmembrane protein 1.870364796 GRAMD4 GRAM domain containing −2.114673101 115 4 DSN1 DSN1 homolog, MIS12 1.868479018 BCL2 BCL2, apoptosis regulator −2.115878669 kinetochore complex component A530099J19RIK Description Not Found 1.867896464 PELI3 pellino E3 ubiquitin protein −2.118915146 ligase family member 3 1700007K09RIK Description Not Found 1.867896464 PPP1CB protein phosphatase 1 −2.119236221 catalytic subunit beta 1810043G02RIK Description Not Found 1.867896464 TFF2 trefoil factor 2 −2.121015401 UCHL1 ubiquitin C-terminal 1.867896464 GCA grancalcin −2.121015401 hydrolase L1 PTCH2 patched 2 1.867896464 LYL1 LYL1, basic helix-loop- −2.121015401 helix family member APBB3 amyloid beta precursor 1.867896464 ATG4B autophagy related 4B −2.121015401 protein binding family B cysteine peptidase member 3 PTER phosphotriesterase related 1.867896464 CCDC102A coiled-coil domain −2.121015401 containing 102A PRKCE protein kinase C epsilon 1.867896464 ATP2A1 ATPase −2.121015401 sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1 PLEKHM3 pleckstrin homology 1.867896464 TERF2 telomeric repeat binding −2.123585568 domain containing M3 factor 2 HIST1H4C histone cluster 1, H4c 1.867896464 LCN5 lipocalin 5(Lcn5) −2.124432612 PLS3 plastin 3 1.867896464 TM6SF1 transmembrane 6 −2.124533495 superfamily member 1 DUSP4 dual specificity 1.867686654 SSBP2 single stranded DNA −2.129283017 phosphatase 4 binding protein 2 SCLY selenocysteine lyase 1.862802277 KRTAP6-2 keratin associated protein 6- −2.137503524 2 RPRD1A regulation of nuclear pre- 1.861777838 CRHBP corticotropin releasing −2.137503524 mRNA domain hormone binding protein containing 1A CCRL2 C-C motif chemokine 1.86175579 TOPBP1 topoisomerase (DNA) II −2.137503524 receptor like 2 binding protein 1 CCT7 chaperonin containing 1.861636037 SLC35A3 solute carrier family 35 −2.137503524 TCP1 subunit 7 member A3 ZFP217 zinc finger protein 1.861097096 CACNB4 calcium voltage-gated −2.137503524 217(Zfp217) channel auxiliary subunit beta 4 ACTN4 actinin alpha 4 1.859689938 TASP1 taspase 1 −2.137503524 KCNA3 potassium voltage-gated 1.859135363 HMBOX1 homeobox containing 1 −2.145313833 channel subfamily A member 3 CUL7 cullin 7 1.858597911 ZFP62 ZFP62 zinc finger protein −2.145677455 LRRC59 leucine rich repeat 1.857543219 PCDHB4 protocadherin beta 4 −2.148666128 containing 59 PHTF2 putative homeodomain 1.855602651 SLC35F3 solute carrier family 35 −2.15120644 transcription factor 2 member F3 KDELC1 KDEL motif containing 1 1.852556218 AW549877 expressed sequence −2.151324826 AW549877(AW549877) SEC24D SEC24 homolog D, 1.8483841 GIMAP9 GTPase, IMAP family −2.152400921 COPII coat complex member 9(Gimap9) component OLFR222 olfactory receptor 1.847996907 ZFP329 zinc finger protein −2.153805336 222(Olfr222) 329(Zfp329) OLFR118 olfactory receptor 1.847996907 KRT74 keratin 74 −2.153805336 118(Olfr118) CASKIN2 CASK interacting protein 1.847996907 REG3A regenerating family −2.153805336 2 member 3 alpha TPK1 thiamin 1.847996907 RAB4A RAB4A, member RAS −2.154308231 pyrophosphokinase 1 oncogene family NOL3 nucleolar protein 3 1.847996907 CECR5 cat eye syndrome −2.155682653 chromosome region, candidate 5 UBA6 ubiquitin like modifier 1.847388943 ESM1 endothelial cell specific −2.157156463 activating enzyme 6 molecule 1 RAVER1 ribonucleoprotein, PTB 1.846151947 HS6ST1 heparan sulfate 6-O- −2.164820712 binding 1 sulfotransferase 1 NAT10 N-acetyltransferase 10 1.843300131 DDB2 damage specific DNA −2.168338824 binding protein 2 HIST1H3H histone cluster 1, H3h 1.842055889 5430435G22RIK Description Not Found −2.169925001 SNX8 sorting nexin 8 1.840985134 ALOX12B arachidonate 12- −2.169925001 lipoxygenase, 12R type POLR3K polymerase (RNA) III 1.839538616 SLC34A3 solute carrier family 34 −2.169925001 subunit K member 3 WDR55 WD repeat domain 55 1.835957408 TNS4 tensin 4 −2.169925001 WDR93 WD repeat domain 93 1.830541464 CANX calnexin −2.169925001 PLSCR1 phospholipid scramblase 1.828635636 BET1 Bet1 golgi vesicular −2.169925001 1 membrane trafficking protein ARL6 ADP ribosylation factor 1.827819025 BEST2 bestrophin 2 −2.169925001 like GTPase 6 NOL9 nucleolar protein 9 1.827819025 USP28 ubiquitin specific peptidase −2.172998154 28 PNKD paroxysmal 1.827819025 PDE4B phosphodiesterase 4B −2.173614018 nonkinesigenic dyskinesia TMEM139 transmembrane protein 1.827819025 CNOT4 CCR4-NOT transcription −2.177917792 139 complex subunit 4 ASPH aspartate beta- 1.827819025 NECAP1 NECAP endocytosis −2.178043245 hydroxylase associated 1 LZTFL1 leucine zipper 1.827819025 JUN Jun proto-oncogene, AP-1 −2.178565309 transcription factor like 1 transcription factor subunit RHEBL1 Ras homolog enriched in 1.827819025 SLC10A7 solute carrier family 10 −2.17990909 brain like 1 member 7 CHCHD5 coiled-coil-helix-coiled- 1.82552849 IL17A interleukin 17A −2.181702586 coil-helix domain containing 5 GPD2 glycerol-3-phosphate 1.824148697 ERICH1 glutamate rich 1 −2.182286216 dehydrogenase 2 STK39 serine/threonine kinase 39 1.823608879 HN1L hematological and −2.185866545 neurological expressed 1- like MAGED2 MAGE family member 1.820863253 SLFNL1 schlafen like 1 −2.185866545 D2 TBC1D9B TBC1 domain family 1.813219568 MYOD1 myogenic differentiation 1 −2.185866545 member 9B LSS lanosterol synthase (2,3- 1.809540228 TRIM35 tripartite motif containing −2.185866545 oxidosqualene-lanosterol 35 cyclase) OLFR859 olfactory receptor 1.807354922 CHRNE cholinergic receptor −2.186397884 859(Olfr859) nicotinic epsilon subunit OLFR1225 olfactory receptor 1.807354922 PHF21A PHD finger protein 21A −2.190943197 1225(Olfr1225) IFNA11 interferon alpha 1.807354922 HIST1H2AE histone cluster 1, H2ae −2.196698179 11(Ifna11) ARG1 arginase 1 1.807354922 SATB1 SATB homeobox 1 −2.198659952 ASCL3 achaete-scute family 1.807354922 LCN8 lipocalin 8 −2.201633861 bHLH transcription factor 3 AGA aspartylglucosaminidase 1.807354922 ABCG5 ATP binding cassette −2.201633861 subfamily G member 5 MAP3K12 mitogen-activated protein 1.806530545 KRBA1 KRAB-A domain −2.202959029 kinase kinase kinase 12 containing 1 COMMD10 COMM domain 1.802771724 CD274 CD274 molecule −2.206081393 containing 10 STYX serine/threonine/tyrosine 1.801251483 DYRK2 dual specificity tyrosine −2.206730511 interacting protein phosphorylation regulated kinase 2 EPHA6 EPH receptor A6 1.797583147 ZFP292 zinc finger protein −2.209453366 292(Zfp292) SERPINA3F serine (or cysteine) 1.794445043 PRX periaxin −2.209453366 peptidase inhibitor, clade A, member 3F(Serpina3f) PUS10 pseudouridylate synthase 1.791814071 SPAG1 sperm associated antigen 1 −2.209453366 10 RASL12 RAS like family 12 1.791652715 ASGR2 asialoglycoprotein receptor −2.209784456 2 MRPL51 mitochondrial ribosomal 1.787631232 PTEN phosphatase and tensin −2.215013513 protein L51 homolog OLFR1306 olfactory receptor 1.786596362 IL1A interleukin 1 alpha −2.217230716 1306(Olfr1306) BCL2A1C B cell 1.786596362 TPCN2 two pore segment channel 2 −2.217230716 leukemia/lymphoma 2 related protein Alc(Bcl2a1c) HOXD1 homeobox D1 1.786596362 IKBKB inhibitor of kappa light −2.217230716 polypeptide gene enhancer in B-cells, kinase beta MEMO1 mediator of cell motility 1 1.786596362 ST6GAL1 ST6 beta-galactoside alpha- −2.218342351 2,6-sialyltransferase 1 ARCN1 archain 1 1.786596362 TMEM161A transmembrane protein −2.232660757 161A NUDT10 nudix hydrolase 10 1.786596362 STK32B serine/threonine kinase 32B −2.232660757 SLC4A4 solute carrier family 4 1.786596362 CHST14 carbohydrate −2.232660757 member 4 sulfotransferase 14 DHRS4 dehydrogenase/reductase 1.786596362 AQP3 aquaporin 3 (Gill blood −2.232660757 4 group) TOM1 target of myb1 membrane 1.786596362 RASSF3 Ras association domain −2.233505898 trafficking protein family member 3 TST thiosulfate 1.786596362 OTUD7B OTU deubiquitinase 7B −2.242923867 sulfurtransferase RIPK2 receptor interacting 1.784428584 AP3M2 adaptor related protein −2.247481244 serine/threonine kinase 2 complex 3 mu 2 subunit NAIP2 NLR family, apoptosis 1.780351745 PSMA6 proteasome subunit alpha 6 −2.247927513 inhibitory protein 2(Naip2) OLFR133 olfactory receptor 1.77946628 PRCC papillary renal cell −2.247927513 133(Olfr133) carcinoma (translocation- associated) NBR1 NBR1, autophagy cargo 1.776995396 ZFP688 zinc finger protein −2.262218541 receptor 688(Zfp688) GLIS1 GLIS family zinc finger 1 1.776512203 DOCK11 dedicator of cytokinesis 11 −2.262218541 SLC35A2 solute carrier family 35 1.776232819 PLA2G4F phospholipase A2 group −2.263034406 member A2 IVF AU022252 expressed sequence 1.774559318 MYPN myopalladin −2.263034406 AU022252(AU022252) OLFR64 olfactory receptor 1.773991786 FRS2 fibroblast growth factor −2.263034406 64(Olfr64) receptor substrate 2 PPAPDC2 Description Not Found 1.771983065 STARD6 StAR related lipid transfer −2.263034406 domain containing 6 DIS3 DIS3 homolog, exosome 1.771375295 WSCD2 WSC domain containing 2 −2.270653766 endoribonuclease and 3′- 5′ exoribonuclease 4931440F15RIK Description Not Found 1.770829046 TLE1 transducin like enhancer of −2.272631746 split 1 ZFP771 zinc finger protein 1.77019569 HDHD3 haloacid dehalogenase like −2.272966802 771(Zfp771) hydrolase domain containing 3 HMBS hydroxymethylbilane 1.769676967 1700029J07RIK Description Not Found −2.277984747 synthase RCC1 regulator of chromosome 1.768267605 CLEC2D C-type lectin domain family −2.277984747 condensation 1 2 member D SPAG5 sperm associated antigen 1.767980257 PPM1G protein phosphatase, −2.277984747 5 Mg2+/Mn2+ dependent 1G TSPAN31 tetraspanin 31 1.767626782 CDKN1B cyclin dependent kinase −2.280970508 inhibitor 1B PCDHGB8 protocadherin gamma 1.765534746 OASL1 2′-5′ oligoadenylate −2.28169825 subfamily B, 8(Pcdhgb8) synthetase-like 1(Oasl1) PRL2B1 prolactin family 2, 1.765534746 G0S2 G0/G1 switch 2 −2.282045463 subfamily b, member 1(Prl2b1) OBOX5 oocyte specific homeobox 1.765534746 TMEM17 transmembrane protein 17 −2.285402219 5(Obox5) PIK3R3 phosphoinositide-3-kinase 1.765534746 BLVRB biliverdin reductase B −2.290619427 regulatory subunit 3 MAP3K4 mitogen-activated protein 1.765534746 GOSR1 golgi SNAP receptor −2.290897209 kinase kinase kinase 4 complex member 1 LRRC30 leucine rich repeat 1.765534746 ZFP26 zinc finger protein −2.292781749 containing 30 26(Zfp26) EN2 engrailed homeobox 2 1.765534746 CXCL2 C-X-C motif chemokine −2.292781749 ligand 2 HOOK3 hook microtubule- 1.765534746 SNX7 sorting nexin 7 −2.292781749 tethering protein 3 MYO9A myosin IXA 1.765534746 ZDHHC23 zinc finger DHHC-type −2.292781749 containing 23 STX7 syntaxin 7 1.765060364 GALNT6 polypeptide N- −2.292781749 acetylgalactosaminyl- transferase 6 ATM ATM serine/threonine 1.763504031 AMPD1 adenosine monophosphate −2.297844157 kinase deaminase 1 KCNK6 potassium two pore 1.763385753 GIMAP5 GTPase, IMAP family −2.303246615 domain channel member 5 subfamily K member 6 PQLC3 PQ loop repeat containing 1.759954577 ATP5F1 ATP synthase, H+ −2.305399163 3 transporting, mitochondrial Fo complex subunit B1 KIFAP3 kinesin associated protein 1.758843168 LHFPL2 lipoma HMGIC fusion −2.307428525 3 partner-like 2 E2F4 E2F transcription factor 4 1.757752886 KIF1B kinesin family member 1B −2.313231129 ETV5 ETS variant 5 1.757709335 TLE6 transducin like enhancer of −2.321928095 split 6 GTF2E2 general transcription 1.75666387 SHF Src homology 2 domain −2.330691998 factor IIE subunit 2 containing F GPR150 G protein-coupled 1.75547927 NGFR nerve growth factor −2.331438521 receptor 150 receptor E130308A19RIK RIKEN cDNA 1.754887502 KLRA4 killer cell lectin-like −2.334485632 E130308A19 receptor, subfamily A, gene(E130308A19Rik) member 4(Klra4) DPYSL4 dihydropyrimidinase like 1.754887502 ITGAE integrin subunit alpha E −2.335948972 4 FNBP1 formin binding protein 1 1.75468902 PQLC2 PQ loop repeat containing 2 −2.336141568 TMOD4 tropomodulin 4 1.754064107 KLRB1A killer cell lectin-like −2.336283388 receptor subfamily B member 1A(Klrb1a) ERLIN1 ER lipid raft associated 1 1.751154691 IRF9 interferon regulatory factor −2.336308285 9 ENOPH1 enolase-phosphatase 1 1.748447442 GATA3 GATA binding protein 3 −2.338971433 RAB31 RAB31, member RAS 1.746215332 RSAD2 radical S-adenosyl −2.33997952 oncogene family methionine domain containing 2 HOXA6 homeobox A6 1.745184623 RNF215 ring finger protein 215 −2.341976415 TAS2R126 taste receptor, type 2, 1.744161096 IL7R interleukin 7 receptor −2.343395577 member 126(Tas2r126) AGXT2 alanine--glyoxylate 1.744161096 ACP5 acid phosphatase 5, tartrate −2.345270806 aminotransferase 2 resistant STK32C serine/threonine kinase 1.744161096 STYXL1 serine/threonine/tyrosine −2.346956889 32C interacting-like 1 P2RY2 purinergic receptor P2Y2 1.744161096 NOXO1 NADPH oxidase organizer −2.35030956 1 NWD1 NACHT and WD repeat 1.744161096 IGFALS insulin like growth factor −2.358664554 domain containing 1 binding protein acid labile subunit UQCRQ ubiquinol-cytochrome c 1.744161096 STIM1 stromal interaction −2.359335599 reductase complex III molecule 1 subunit VII PPP1R3A protein phosphatase 1 1.744161096 TMEM186 transmembrane protein 186 −2.361030771 regulatory subunit 3A GOLT1A golgi transport 1A 1.744161096 OLFR1043 olfactory receptor −2.364572432 1043(Olfr1043) EZH1 enhancer of zeste 1 1.744161096 D8ERTD82E DNA segment, Chr 8, −2.364572432 polycomb repressive ERATO Doi 82, complex 2 subunit expressed(D8Ertd82e) MTHFD2 methylenetetrahydrofolate 1.744154314 MYOG myogenin −2.364572432 dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase PGRMC1 progesterone receptor 1.742545062 NCLN nicalin −2.364572432 membrane component 1 DNAJB12 DnaJ heat shock protein 1.741863621 MTSS1 metastasis suppressor 1 −2.364572432 family (Hsp40) member B12 DNAJC11 DnaJ heat shock protein 1.738767837 TRMU tRNA 5- −2.364572432 family (Hsp40) member methylaminomethyl-2- C11 thiouridylate methyltransferase TOMM6 translocase of outer 1.738448709 EMILIN2 elastin microfibril interfacer −2.369119767 mitochondrial membrane 2 6 RPS6KL1 ribosomal protein S6 1.738393453 MPV17L MPV17 mitochondrial inner −2.371558863 kinase like 1 membrane protein like CDC73 cell division cycle 73 1.73665741 WWC2 WW and C2 domain −2.371558863 containing 2 NDC80 NDC80, kinetochore 1.732078892 TMEM178 transmembrane protein −2.374005585 complex component 178(Tmem178) TACC3 transforming acidic 1.731372884 TPCN1 two pore segment channel 1 −2.375232208 coiled-coil containing protein 3 CPSF3 cleavage and 1.727926568 LRRC45 leucine rich repeat −2.377207351 polyadenylation specific containing 45 factor 3 ARID3A AT-rich interaction 1.726471722 1110059G10RIK Description Not Found −2.377915929 domain 3A LLPH LLP homolog, long-term 1.726107859 MCOLN2 mucolipin 2 −2.378511623 synaptic facilitation PCNA proliferating cell nuclear 1.725441599 DDX58 DEXD/H-box helicase 58 −2.378511623 antigen GJC2 gap junction protein 1.722978517 H2-OA histocompatibility 2, O −2.382329516 gamma 2 region alpha locus(H2-Oa) OLFR373 olfactory receptor 1.722466024 RARG retinoic acid receptor −2.388827772 373(Olfr373) gamma H2-T24 histocompatibility 2, T 1.722466024 SERPINB1A serine (or cysteine) −2.392317423 region locus 24(H2-T24) peptidase inhibitor, clade B, member la(Serpinb1a) AKAP7 A-kinase anchoring 1.722466024 GHRL ghrelin/obestatin −2.392317423 protein 7 prepropeptide NDUFB7 NADH:ubiquinone 1.722466024 ZMAT4 zinc finger matrin-type 4 −2.392317423 oxidoreductase subunit B7 PRR11 proline rich 11 1.722466024 BTBD6 BTB domain containing 6 −2.392897478 TJP1 tight junction protein 1 1.722466024 KLRA16 killer cell lectin-like −2.394534969 receptor, subfamily A, member 16(Klra16) S100A3 S100 calcium binding 1.722466024 EPS15L1 epidermal growth factor −2.397012831 protein A3 receptor pathway substrate 15 like 1 KRT78 keratin 78 1.718729711 VCPIP1 valosin containing protein −2.397303585 interacting protein 1 GMDS GDP-mannose 4,6- 1.717904741 RRP7A ribosomal RNA processing −2.404992223 dehydratase 7 homolog A PDGFB platelet derived growth 1.714400534 IL1B interleukin 1 beta −2.40599236 factor subunit B SLC36A1 solute carrier family 36 1.714297338 NAT14 N-acetyltransferase 14 −2.40599236 member 1 (putative) RSU1 Ras suppressor protein 1 1.712647036 SLC40A1 solute carrier family 40 −2.40599236 member 1 STX12 syntaxin 12 1.711911478 RAB37 RAB37, member RAS −2.40599236 oncogene family SLC25A34 solute carrier family 25 1.711494907 IL 17RA interleukin 17 receptor A −2.40599236 member 34 AFG3L2 AFG3 like matrix AAA 1.711057 BACE1 beta-secretase 1 −2.40599236 peptidase subunit 2 RPL24 ribosomal protein L24 1.709193708 CTNS cystinosin, lysosomal −2.40599236 cystine transporter UBE3C ubiquitin protein ligase 1.708789682 IFIT3 interferon induced protein −2.411404504 E3C with tetratricopeptide repeats 3 CAR12 carbonic anhydrase 1.70867626 ZFYVE21 zinc finger FYVE-type −2.412378292 12(Car12) containing 21 ZFP207 zinc finger protein 1.707603009 1700016D06RIK Description Not Found −2.419538892 207(Zfp207) XIST X inactive specific 1.706065607 STK25 serine/threonine kinase 25 −2.419538892 transcript (non-protein coding) NCAPD2 non-SMC condensin I 1.705012178 PLEKHJ1 pleckstrin homology −2.419538892 complex subunit D2 domain containing J1 ZSWIM2 zinc finger SWIM-type 1.704802998 TGIF2 TGFB induced factor −2.419538892 containing 2 homeobox 2 CASP1 caspase 1 1.70065942 SLC25A29 solute carrier family 25 −2.419538892 member 29 OLFR701 olfactory receptor 1.700439718 DAPL1 death associated protein like −2.419661316 701(Olfr701) 1 CBLC Cbl proto-oncogene C 1.700439718 P2RX4 purinergic receptor P2X 4 −2.425748008 HIST1H2AC histone cluster 1, H2ac 1.700439718 1700001O22RIK Description Not Found −2.426264755 EPHA10 EPH receptor A10 1.700439718 C9 complement component 9 −2.429615964 NDUFC2 NADH:ubiquinone 1.700439718 KLF13 Kruppel like factor 13 −2.430628023 oxidoreductase subunit C2 DLG1 discs large MAGUK 1.700439718 GADD45A growth arrest and DNA −2.432591239 scaffold protein 1 damage inducible alpha SCN10A sodium voltage-gated 1.700439718 OLFR788 olfactory receptor −2.432959407 channel alpha subunit 10 788(Olfr788) RGL3 ral guanine nucleotide 1.700439718 FADS6 fatty acid desaturase 6 −2.432959407 dissociation stimulator like 3 TMCO3 transmembrane and 1.700439718 CHCHD2 coiled-coil-helix-coiled- −2.432959407 coiled-coil domains 3 coil-helix domain containing 2 BCL2L14 BCL2 like 14 1.700439718 MPPE1 metallophosphoesterase 1 −2.432959407 THOP1 thimet oligopeptidase 1 1.700290033 CHAC1 ChaC glutathione specific −2.432959407 gamma- glutamylcyclotransferase 1 MTIF3 mitochondrial 1.698305331 2310011J03RIK Description Not Found −2.435017448 translational initiation factor 3 XDH xanthine dehydrogenase 1.697717724 LRSAM1 leucine rich repeat and −2.437473925 sterile alpha motif containing 1 ANXA9 annexin A9 1.697184071 SIRPA signal regulatory protein −2.443125132 alpha OLFR1502 olfactory receptor 1.694046727 CYP24A1 cytochrome P450 family 24 −2.44625623 1502(Olfr1502) subfamily A member 1 HCFC2 host cell factor C2 1.693780609 NQO1 NAD(P)H quinone −2.44625623 dehydrogenase 1 DIDO1 death inducer-obliterator 1.693596948 HRH4 histamine receptor H4 −2.44625623 1 PGAM1 phosphoglycerate mutase 1.689846917 NUDCD1 NudC domain containing 1 −2.44625623 1 RASGEFIC RasGEF domain family 1.689299161 CCND1 cyclin D1 −2.447924527 member 1C SLC25A42 solute carrier family 25 1.686774817 ADAM22 ADAM metallopeptidase −2.452858965 member 42 domain 22 CPT2 carnitine 1.686364794 MDK midkine (neurite growth- −2.456149035 palmitoyltransferase 2 promoting factor 2) MAD2L1 MAD2 mitotic arrest 1.686161103 STX1A syntaxin 1A −2.456729828 deficient-like 1 (yeast) NQO2 NAD(P)H quinone 1.685558757 HEMK1 HemK methyltransferase −2.459431619 dehydrogenase 2 family member 1 HIP1R huntingtin interacting 1.685473307 B4GALT7 beta-1,4- −2.459431619 protein 1 related galactosyltransferase 7 ALOX12E arachidonate 1.684373244 ASXL2 additional sex combs like 2, −2.459431619 lipoxygenase, transcriptional regulator epidermal(Alox12e) LMAN1 lectin, mannose binding 1 1.683514205 TLR7 toll like receptor 7 −2.46052038 ASB3 ankyrin repeat and SOCS 1.680142991 TDP1 tyrosyl-DNA −2.464461869 box containing 3 phosphodiesterase 1 XKR5 XK related 5 1.679254438 1700025G04RIK Description Not Found −2.469303076 ZFP235 zinc finger protein 1.678071905 SLC16A6 solute carrier family 16 −2.471045434 235(Zfp235) member 6 OLFR971 olfactory receptor 1.678071905 DOXL2 diamine oxidase-like −2.472487771 971(Olfr971) protein 2(Dox12) OLFR374 olfactory receptor 1.678071905 PKD1L3 polycystin 1 like 3, transient −2.472487771 374(Olfr374) receptor potential channel interacting NOS1AP nitric oxide synthase 1 1.678071905 ZC3H11A zinc finger CCCH-type −2.472487771 adaptor protein containing 11A GALM galactose mutarotase 1.678071905 LY6K lymphocyte antigen 6 −2.472487771 complex, locus K MEGF9 multiple EGF like 1.678071905 KLF7 Kruppel like factor 7 −2.474755307 domains 9 CCDC66 coiled-coil domain 1.678071905 BTLA B and T lymphocyte −2.475604026 containing 66 associated LRRC40 leucine rich repeat 1.678071905 CDON cell adhesion associated, −2.485426827 containing 40 oncogene regulated RALA RALA Ras like proto- 1.678071905 DDC dopa decarboxylase −2.485426827 oncogene A YIPF4 Yip1 domain family 1.678071905 GTF2A2 general transcription factor −2.485426827 member 4 IIA subunit 2 TAL2 T-cell acute lymphocytic 1.678071905 DTX4 deltex E3 ubiquitin ligase 4 −2.485426827 leukemia 2 LRRC8A leucine rich repeat 1.678071905 GSTK1 glutathione S-transferase −2.486195934 containing 8 family kappa 1 member A APOM apolipoprotein M 1.678071905 OLFR213 olfactory receptor −2.489125048 213(Olfr213) KCNG3 potassium voltage-gated 1.678071905 PDE5A phosphodiesterase 5A −2.490571469 channel modifier subfamily G member 3 CNN1 calponin 1 1.678071905 TOB1 transducer of ERBB2, 1 −2.496763907 STAC2 SH3 and cysteine rich 1.678071905 1700109H08RIK Description Not Found −2.498250868 domain 2 SFRP2 secreted frizzled related 1.678071905 LEFTY1 left-right determination −2.498250868 protein 2 factor 1 SERPINB9E serine (or cysteine) 1.670169131 SNAPC4 small nuclear RNA −2.500878922 peptidase inhibitor, clade activating complex B, member 9e(Serpinb9e) polypeptide 4 TFB1M transcription factor B1, 1.668946692 RNF41 ring finger protein 41 −2.503551585 mitochondrial SLC25A10 solute carrier family 25 1.668856925 KLHL34 kelch like family member −2.504620392 member 10 34 BID BH3 interacting domain 1.667992567 SSH2 slingshot protein −2.505492762 death agonist phosphatase 2 MRPS27 mitochondrial ribosomal 1.667295766 CAMK2B calcium/calmodulin −2.507047355 protein S27 dependent protein kinase II beta NEDD4 neural precursor cell 1.666756592 IRF7 interferon regulatory factor −2.507590939 expressed, 7 developmentally down- regulated 4, E3 ubiquitin protein ligase VANGL2 VANGL planar cell 1.666756592 SCML4 sex comb on midleg-like 4 −2.523118672 polarity protein 2 (Drosophila) UBE2R2 ubiquitin conjugating 1.666641116 EPB4.1 Description Not Found −2.523561956 enzyme E2 R2 KLHL30 kelch like family member 1.666519523 PARP12 poly(ADP-ribose) −2.523561956 30 polymerase family member 12 FBXO36 F-box protein 36 1.665588375 CACNB3 calcium voltage-gated −2.529877218 channel auxiliary subunit beta 3 DCT dopachrome tautomerase 1.664016818 NRG4 neuregulin 4 −2.53318567 CCDC120 coiled-coil domain 1.663931727 OLFR1383 olfactory receptor −2.5360529 containing 120 1383(Olfr1383) TMEM38B transmembrane protein 1.663455268 PTGR1 prostaglandin reductase 1 −2.5360529 38B ENDOD1 endonuclease domain 1.663327923 NFAM1 NFAT activating protein −2.5360529 containing 1 with ITAM motif 1 PTPRD protein tyrosine 1.663215776 ARL4C ADP ribosylation factor like −2.5360529 phosphatase, receptor GTPase 4C type D ARL3 ADP ribosylation factor 1.661690196 LACE1 lactation elevated 1 −2.5360529 like GTPase 3 CDC37 cell division cycle 37 1.661567827 CDC14B cell division cycle 14B −2.545350645 MKKS McKusick-Kaufman 1.66106548 GUCA1A guanylate cyclase activator −2.548436625 syndrome 1A CHN2 chimerin 2 1.660998764 KIF21B kinesin family member 21B −2.554588852 CRTAP cartilage associated 1.659431912 ARID3B AT-rich interaction domain −2.558087884 protein 3B CXCR6 C-X-C motif chemokine 1.657515938 HBA-A1 hemoglobin alpha, adult −2.560714954 receptor 6 chain 1(Hba-a1) BUB1B BUB1 mitotic checkpoint 1.65691495 CSF2RB2 colony stimulating factor 2 −2.560714954 serine/threonine kinase B receptor, beta 2, low- affinity (granulocyte- macrophage)(Csf2rb2) B430306N03RIK RIKEN cDNA 1.655351829 ATP6V1B1 ATPase H+ transporting V1 −2.560714954 B430306N03 subunit B1 gene(B430306N03Rik) OLFR1262 olfactory receptor 1.655351829 PCSK1N proprotein convertase −2.560714954 1262(Olfr1262) subtilisin/kexin type 1 inhibitor SLC38A5 solute carrier family 38 1.655351829 ZFP667 zinc finger protein −2.566670372 member 5 667(Zfp667) VAT1L vesicle amine transport 1- 1.655351829 SH3BP1 SH3 domain binding −2.566734604 like protein 1 HOXB7 homeobox B7 1.655351829 FFAR2 free fatty acid receptor 2 −2.572889668 GAN gigaxonin 1.655351829 EEF2K eukaryotic elongation factor −2.572889668 2 kinase MMP28 matrix metallopeptidase 1.655351829 SLPI secretory leukocyte −2.574721828 28 peptidase inhibitor METTL10 methyltransferase like 10 1.655351829 CMA1 chymase 1 −2.584962501 SIX4 SIX homeobox 4 1.655351829 ASCL1 achaete-scute family bHLH −2.584962501 transcription factor 1 TDRD6 tudor domain containing 6 1.655351829 ACPP acid phosphatase, prostate −2.584962501 COMMD5 COMM domain 1.654604999 CLCNKB chloride voltage-gated −2.596935142 containing 5 channel Kb PRDX4 peroxiredoxin 4 1.651923925 FBXW7 F-box and WD repeat −2.596935142 domain containing 7 HS3ST3A1 heparan sulfate- 1.649298274 OLIG3 oligodendrocyte −2.596935142 glucosamine 3- transcription factor 3 sulfotransferase 3A1 CALCA calcitonin related 1.649067786 WHRN whirlin −2.606789951 polypeptide alpha SLC12A2 solute carrier family 12 1.648449243 DNAJC14 DnaJ heat shock protein −2.608809243 member 2 family (Hsp40) member C14 TJP2 tight junction protein 2 1.644145647 PIGT phosphatidylinositol glycan −2.611031218 anchor biosynthesis class T LRRC16B Description Not Found 1.64385619 AP1G2 adaptor related protein −2.614709844 complex 1 gamma 2 subunit AP3S2 adaptor related protein 1.64385619 SAA2 serum amyloid A2 −2.62058641 complex 3 sigma 2 subunit PSMD9 proteasome 26S subunit, 1.64385619 USP30 ubiquitin specific peptidase −2.62058641 non-ATPase 9 30 PARD6G par-6 family cell polarity 1.643379419 RPE65 retinal pigment epithelium −2.632268216 regulator gamma specific protein 65 CIAPIN1 cytokine induced 1.643219709 CML1 Description Not Found −2.634891632 apoptosis inhibitor 1 CKAP5 cytoskeleton associated 1.642747156 SLC6A19 solute carrier family 6 −2.640930751 protein 5 member 19 E430025E21RIK RIKEN cDNA 1.641902626 FGF15 fibroblast growth factor −2.64385619 E430025E21 15(Fgf15) gene(E430025E21Rik) PIAS3 protein inhibitor of 1.641884484 HERC3 HECT and RLD domain −2.64385619 activated STAT 3 containing E3 ubiquitin protein ligase 3 USP1 ubiquitin specific 1.640233791 ADAMTSL4 ADAMTS like 4 −2.64385619 peptidase 1 RAB3GAP2 RAB3 GTPase activating 1.639592623 HYAL3 hyaluronoglucosaminidase −2.64385619 non-catalytic protein 3 subunit 2 CSRP2 cysteine and glycine rich 1.639046229 SLC15A2 solute carrier family 15 −2.648217996 protein 2 member 2 MOV10 Mov10 RISC complex 1.638073837 UFSP1 UFM1-specific peptidase 1 −2.649553823 RNA helicase (inactive) GM1965 predicted gene 1.637881562 6430573F11RIK Description Not Found −2.655351829 1965(Gm1965) POMGNT1 protein O-linked mannose 1.636237884 DNM3OS DNM3 opposite −2.655351829 N- strand/antisense RNA acetylglucosaminyltransfe rase 1 (beta 1,2-) FIGNL1 fidgetin like 1 1.633950492 F2RL1 F2R like trypsin receptor 1 −2.655351829 TMEM177 transmembrane protein 1.633475547 SNX33 sorting nexin 33 −2.666654581 177 ALX4 ALX homeobox 4 1.632864872 CXCL9 C-X-C motif chemokine −2.666756592 ligand 9 OLFR533 olfactory receptor 1.632268216 TEAD2 TEA domain transcription −2.666756592 533(Olfr533) factor 2 H2-M10.3 histocompatibility 2, M 1.632268216 QSOX1 quiescin sulfhydryl oxidase −2.666756592 region locus 10.3(H2- 1 M10.3) GPX7 glutathione peroxidase 7 1.632268216 TLR13 toll-like receptor 13(Tlr13) −2.678071905 STXBP6 syntaxin binding protein 6 1.632268216 SCD3 stearoyl-coenzyme A −2.678071905 desaturase 3(Scd3) RAB33A RAB33A, member RAS 1.632268216 SDC3 syndecan 3 −2.678071905 oncogene family PDCL3 phosducin like 3 1.632268216 GRPR gastrin releasing peptide −2.678071905 receptor GPR20 G protein-coupled 1.632268216 MAFK MAF bZIP transcription −2.678071905 receptor 20 factor K GSTA2 glutathione S-transferase 1.632268216 DIRC2 disrupted in renal −2.678071905 alpha 2 carcinoma 2 ADCY10 adenylate cyclase 10 1.632268216 ZCCHC12 zinc finger CCHC-type −2.67833354 (soluble) containing 12 PEX12 peroxisomal biogenesis 1.632268216 ADCY6 adenylate cyclase 6 −2.680886921 factor 12 IQCC IQ motif containing C 1.632268216 ECM1 extracellular matrix protein −2.68345512 1 ENPP1 ectonucleotide 1.632086279 AFP alpha fetoprotein −2.689299161 pyrophosphatase/ phosphodiesterase 1 ADAL adenosine deaminase-like 1.630664126 GP5 glycoprotein V platelet −2.689299161 SCRN2 secernin 2 1.630566247 GAB3 GRB2 associated binding −2.691405681 protein 3 CEP78 centrosomal protein 78 1.629851642 USP2 ubiquitin specific peptidase −2.693334369 2 SLC25A15 solute carrier family 25 1.629798606 PLXNB1 plexin B1 −2.700439718 member 15 ADSSL1 adenylosuccinate synthase 1.628272149 PODXL2 podocalyxin like 2 −2.700799925 like 1 TM6SF2 transmembrane 6 1.627758638 RAD9B RAD9 checkpoint clamp −2.70103836 superfamily member 2 component B TUBG1 tubulin gamma 1 1.624511879 AKAP10 A-kinase anchoring protein −2.705977902 10 FASTK Fas activated 1.623336662 PIGW phosphatidylinositol glycan −2.716990894 serine/threonine kinase anchor biosynthesis class W RBBP5 RB binding protein 5, 1.622163711 COL12A1 collagen type XII alpha 1 −2.722466024 histone lysine chain methyltransferase complex subunit 1700071K01RIK Description Not Found 1.621465074 GPR137B G protein-coupled receptor −2.733354341 137B SLC25A33 solute carrier family 25 1.621282718 IMMP2L inner mitochondrial −2.733354341 member 33 membrane peptidase subunit 2 MDM4 MDM4, p53 regulator 1.62058641 PIK3CB phosphatidylinositol-4,5- −2.737320423 bisphosphate 3-kinase catalytic subunit beta TOP2A topoisomerase (DNA) II 1.620374948 TGFBI transforming growth factor −2.740276443 alpha beta induced OLFR139 olfactory receptor 1.619731323 ZFP106 zinc finger protein −2.744161096 139(Olfr139) 106(Zfp106) PAPLN papilin, proteoglycan like 1.618762248 ARNTL aryl hydrocarbon receptor −2.744161096 sulfated glycoprotein nuclear translocator like PACSIN2 protein kinase C and 1.617401771 HS3ST3B1 heparan sulfate- −2.744161096 casein kinase substrate in glucosamine 3- neurons 2 sulfotransferase 3B1 TRDMT1 tRNA aspartic acid 1.615239219 OASL2 2′-5′ oligoadenylate −2.744892108 methyltransferase 1 synthetase-like 2(Oas12) 4932438H23RIK Description Not Found 1.614681809 PRDX6 peroxiredoxin 6 −2.75084462 SPAG9 sperm associated antigen 1.614567709 RASA2 RAS p21 protein activator 2 −2.751203108 9 RPA3 replication protein A3 1.61436984 HOXB2 homeobox B2 −2.754887502 GNPTAB N-acetylglucosamine-1- 1.613298199 TULP3 tubby like protein 3 −2.754887502 phosphate transferase alpha and beta subunits SNX9 sorting nexin 9 1.609251493 MFRP membrane frizzled-related −2.754887502 protein OLFR550 olfactory receptor 1.609195813 MEN1 menin 1 −2.757556689 550(Olfr550) ZFP160 zinc finger protein 1.608809243 C330021F23RIK RIKEN cDNA −2.762199201 160(Zfp160) C330021F23 gene(C330021F23Rik) TAS2R129 taste receptor, type 2, 1.608809243 CSTAD CSA-conditional, T cell −2.765534746 member 129(Tas2r129) activation-dependent protein(Cstad) OLFR371 olfactory receptor 1.608809243 ALDH5A1 aldehyde dehydrogenase 5 −2.773022439 371(Olfr371) family member A1 OLFR281 olfactory receptor 1.608809243 EPM2AIP1 EPM2A interacting protein −2.773468928 281(Olfr281) 1 OLFR195 olfactory receptor 1.608809243 PDE8B phosphodiesterase 8B −2.776103988 195(Olfr195) OLFR142 olfactory receptor 1.608809243 DMRTA1 DMRT like family A1 −2.776184379 142(Olfr142) PRSS3 protease, serine 3 1.608809243 LYPD6B LY6/PLAUR domain −2.780048768 containing 6B CX3CL1 C-X3-C motif chemokine 1.608809243 CD300E CD300e molecule −2.786596362 ligand 1 TMPRSS6 transmembrane protease, 1.608809243 NPFF neuropeptide FF-amide −2.786596362 serine 6 peptide precursor ALK anaplastic lymphoma 1.608809243 FASTKD1 FAST kinase domains 1 −2.793765229 receptor tyrosine kinase ITGA9 integrin subunit alpha 9 1.608809243 OLFR802 olfactory receptor −2.797012978 802(Olfr802) TIMM13 translocase of inner 1.608809243 HIVEP1 human immunodeficiency −2.797012978 mitochondrial membrane virus type I enhancer 13 binding protein 1 MSH5 mutS homolog 5 1.608809243 HIC1 hypermethylated in cancer 1 −2.797012978 XPO4 exportin 4 1.605818241 TRIM33 tripartite motif containing −2.802009226 33 MED21 mediator complex subunit 1.603309406 SELL selectin L −2.803274253 CHST12 carbohydrate 1.602612589 EPHX1 epoxide hydrolase 1 −2.803758579 sulfotransferase 12 6030408B16RIK Description Not Found 1.602195565 BCL9 B-cell CLL/lymphoma 9 −2.807354922 SLU7 SLU7 homolog, splicing 1.601548066 STAT2 signal transducer and −2.808521822 factor activator of transcription 2 CDK5RAP2 CDK5 regulatory subunit 1.601120229 ELMO3 engulfment and cell motility −2.812498225 associated protein 2 3 CASP7 caspase 7 1.6002402 HDC histidine decarboxylase −2.815167456 KIF22 kinesin family member 22 1.599011705 AI317395 Description Not Found −2.817623258 E2F1 E2F transcription factor 1 1.598449678 RPL14 ribosomal protein L14 −2.817623258 MXI1 MAX interactor 1, 1.597690116 SNAI1 snail family transcriptional −2.818256244 dimerization protein repressor 1 DONSON downstream neighbor of 1.596935142 NUPR1 nuclear protein 1, −2.827819025 SON transcriptional regulator TBX22 T-box 22 1.596935142 IGSF8 immunoglobulin −2.827819025 superfamily member 8 INPPL1 inositol polyphosphate 1.596300192 SLC12A7 solute carrier family 12 −2.827819025 phosphatase like 1 member 7 CSE1L chromosome segregation 1.59586273 RENBP renin binding protein −2.837431463 1 like NDFIP2 Nedd4 family interacting 1.594709608 ZFP553 zinc finger protein −2.837943242 protein 2 553(Zfp553) LYPD6 LY6/PLAUR domain 1.592962293 LRFN2 leucine rich repeat and −2.837943242 containing 6 fibronectin type III domain containing 2 DDX49 DEAD-box helicase 49 1.592190323 HP haptoglobin −2.839737506 MGLL monoglyceride lipase 1.590948822 TOMM40 translocase of outer −2.847996907 mitochondrial membrane 40 NR4A3 nuclear receptor 1.59092994 GABARAPL2 GABA type A receptor −2.847996907 subfamily 4 group A associated protein like 2 member 3 LRRN3 leucine rich repeat 1.590360181 TMEM86A transmembrane protein 86A −2.855497819 neuronal 3 PTPRK protein tyrosine 1.587927102 LRP1 LDL receptor related −2.857980995 phosphatase, receptor protein 1 type K OLFR1212 olfactory receptor 1.584962501 ATXN1 ataxin 1 −2.857980995 1212(Olfr1212) KLHL2 kelch like family member 1.584962501 FAS Fas cell surface death −2.861524641 2 receptor UBE2G2 ubiquitin conjugating 1.584962501 ZDHHC18 zinc finger DHHC-type −2.882740655 enzyme E2 G2 containing 18 GRIN2A glutamate ionotropic 1.584962501 LARGE Description Not Found −2.887525271 receptor NMDA type subunit 2A INHA inhibin alpha subunit 1.584962501 SP5 Sp5 transcription factor −2.887525271 RNPC3 RNA binding region 1.584962501 ATG7 autophagy related 7 −2.895440528 (RNP1, RRM) containing 3 XKR7 XK related 7 1.584962501 DNAJC27 DnaJ heat shock protein −2.897240426 family (Hsp40) member C27 STX19 syntaxin 19 1.584962501 PCSK4 proprotein convertase −2.900866808 subtilisin/kexin type 4 SLC5A5 solute carrier family 5 1.584962501 RNF141 ring finger protein 141 −2.902073579 member 5 VPS37C VPS37C, ESCRT-I 1.584022655 GRAP2 GRB2-related adaptor −2.904150467 subunit protein 2 ERMP1 endoplasmic reticulum 1.582531434 VIPR1 vasoactive intestinal peptide −2.904484098 metallopeptidase 1 receptor 1 ZFP790 zinc finger protein 1.581046002 CAR15 carbonic anhydrase −2.906890596 790(Zfp790) 15(Car15) AA467197 expressed sequence 1.579947242 RELL2 RELT like 2 −2.906890596 AA467197(AA467197) UBE2Z ubiquitin conjugating 1.57976541 HECA hdc homolog, cell cycle −2.916545968 enzyme E2 Z regulator SOAT2 sterol O-acyltransferase 2 1.577460518 DPM1 dolichyl-phosphate −2.933100475 mannosyltransferase polypeptide 1, catalytic subunit ZMAT5 zinc finger matrin-type 5 1.576986214 AOC2 amine oxidase, copper −2.936235748 containing 2 CDCA3 cell division cycle 1.576323153 HIST2H2BE histone cluster 2, H2be −2.936320631 associated 3 NEUROD2 neuronal differentiation 2 1.576266476 ACAD10 acyl-CoA dehydrogenase −2.942514505 family member 10 WDR35 WD repeat domain 35 1.576120636 NT5E 5′-nucleotidase ecto −2.944039663 TWSG1 twisted gastrulation BMP 1.5758728 SSH1 slingshot protein −2.944858446 signaling modulator 1 phosphatase 1 PPT1 palmitoyl-protein 1.575321868 SEMA4F ssemaphorin 4F −2.948329995 thioesterase 1 IRF8 interferon regulatory 1.574489283 NKD2 naked cuticle homolog 2 −2.953960396 factor 8 PLEKHG5 pleckstrin homology and 1.574066379 TCEB3 transcription elongation −2.95419631 RhoGEF domain factor B subunit 3 containing G5 CDC20 cell division cycle 20 1.573718243 HDAC4 histone deacetylase 4 −2.95419631 MFI2 antigen p97 (melanoma 1.572889668 PCNX pecanex homolog −2.972692654 associated) identified by (Drosophila)(Pcnx) monoclonal antibodies 133.2 and 96.5(Mfi2) HDAC9 histone deacetylase 9 1.571625208 ARL5C ADP ribosylation factor like −2.972692654 GTPase 5C ASF1B anti-silencing function 1B 1.570544039 1600014C10RIK Description Not Found −2.981852653 histone chaperone B3GNT1 Description Not Found 1.569171715 ANKRD23 ankyrin repeat domain 23 −2.981852653 SLC25A14 solute carrier family 25 1.569127395 CLOCK clock circadian regulator −2.985543793 member 14 FYN FYN proto-oncogene, Src 1.567462919 SFI1 SFI1 centrin binding protein −2.986410935 family tyrosine kinase SERPINB6B serine (or cysteine) 1.567348435 HEY1 hes related family bHLH −2.987632559 peptidase inhibitor, clade transcription factor with B, member 6b(Serpinb6b) YRPW motif 1 TOP1MT topoisomerase (DNA) I, 1.567180597 ATP11C ATPase phospholipid −2.99095486 mitochondrial transporting 11C CCDC50 coiled-coil domain 1.566273906 NUDCD3 NudC domain containing 3 −3 containing 50 ZFP414 zinc finger protein 1.565776574 CDC25A cell division cycle 25A −3.000238201 414(Zfp414) OGFOD2 2-oxoglutarate and iron 1.565512016 OLFR135 olfactory receptor −3.017921908 dependent oxygenase 135(Olfr135) domain containing 2 CTNNAL1 catenin alpha like 1 1.563586461 RC3H1 ring finger and CCCH-type −3.019621529 domains 1 CREB3L2 CAMP responsive element 1.561361122 NSG2 neuron specific gene family −3.020466888 binding protein 3 like 2 member 2(Nsg2) OLFR492 olfactory receptor 1.560714954 ID1 inhibitor of DNA binding 1, −3.026800059 492(Olfr492) HLH protein OLFR1312 olfactory receptor 1.560714954 CYP2D22 cytochrome P450, family 2, −3.044282215 1312(Olfr1312) subfamily d, polypeptide 22(Cyp2d22) UPK2 uroplakin 2 1.560714954 H2AFJ H2A histone family −3.044297135 member J RESP18 regulated endocrine 1.560714954 TGFBR3 transforming growth factor −3.053111336 specific protein 18 beta receptor 3 CRCT1 cysteine rich C-terminal 1 1.560714954 IRS2 insulin receptor substrate 2 −3.061776198 NEUROD4 neuronal differentiation 4 1.560714954 ADCY7 adenylate cyclase 7 −3.06608919 SENP1 SUMO1/sentrin specific 1.560714954 HYI hydroxypyruvate isomerase −3.072315809 peptidase 1 (putative) MR1 major histocompatibility 1.560714954 TRIP4 thyroid hormone receptor −3.078951341 complex, class I-related interactor 4 BIVM basic, immunoglobulin- 1.560714954 D730001G18RIK RIKEN cDNA −3.087462841 like variable motif D730001G18 containing gene(D730001G18Rik) KPNA2 karyopherin subunit alpha 1.560714954 PRR7 proline rich 7 (synaptic) −3.087462841 2 BAG2 BCL2 associated 1.560714954 GFPT2 glutamine-fructose-6- −3.09592442 athanogene 2 phosphate transaminase 2 SLC12A8 solute carrier family 12 1.560714954 SCMH1 sex comb on midleg −3.100136671 member 8 homolog 1 (Drosophila) SCN7A sodium voltage-gated 1.560714954 ANKRD12 ankyrin repeat domain 12 −3.107456458 channel alpha subunit 7 SLC5A7 solute carrier family 5 1.560714954 PTPRV protein tyrosine −3.112700133 member 7 phosphatase, receptor type, V(Ptprv) ENPEP glutamyl aminopeptidase 1.560714954 TMEM135 transmembrane protein 135 −3.112700133 ANGPTL4 angiopoietin like 4 1.56060777 AKAP3 A-kinase anchoring protein −3.11460665 3 OSBPL3 oxysterol binding protein 1.559778376 CBR2 carbonyl reductase 2(Cbr2) −3.129283017 like 3 MCFD2 multiple coagulation 1.559617874 CXCL16 C-X-C motif chemokine −3.129283017 factor deficiency 2 ligand 16 MAP2K1 mitogen-activated protein 1.558556708 MBTD1 mbt domain containing 1 −3.145677455 kinase kinase 1 ING2 inhibitor of growth family 1.557223521 UBE2J2 ubiquitin conjugating −3.161887682 member 2 enzyme E2 J2 CDCA5 cell division cycle 1.55643411 STK36 serine/threonine kinase 36 −3.161887682 associated 5 MAP3K7 mitogen-activated protein 1.554463905 SLC14A1 solute carrier family 14 −3.16922072 kinase kinase kinase 7 member 1 (Kidd blood group) GSTT3 glutathione S-transferase, 1.55048277 CTSE cathepsin E −3.177917792 theta 3(Gstt3) PFN2 profilin 2 1.549690793 HSD3B7 hydroxy-delta-5-steroid −3.177917792 dehydrogenase, 3 beta- and steroid delta-isomerase 7 HPS4 HPS4, biogenesis of 1.549115647 3010003L21RIK Description Not Found −3.179249632 lysosomal organelles complex 3 subunit 2 CAPN8 calpain 8 1.548436625 BAI1 Description Not Found −3.186461055 RAB11FIP5 RAB11 family interacting 1.548436625 ZFP451 zinc finger protein −3.187711618 protein 5 451(Zfp451) CD9 CD9 molecule 1.548429184 CCDC28B coiled-coil domain −3.192207249 containing 28B CCR6 C-C motif chemokine 1.548250633 MCF2L MCF.2 cell line derived −3.199672345 receptor 6 transforming sequence like ALG2 ALG2, alpha-1,3/1,6- 1.547992668 BCL6 B-cell CLL/lymphoma 6 −3.201024389 mannosyltransferase BCDIN3D BCDIN3 domain 1.546046129 PFKFB4 6-phosphofructo-2- −3.204935584 containing RNA kinase/fructose-2,6- methyltransferase biphosphatase 4 NT5DC3 5′-nucleotidase domain 1.54522349 PROS1 protein S (alpha) −3.209453366 containing 3 DNAJC18 DnaJ heat shock protein 1.544626916 CTSH cathepsin H −3.21628737 family (Hsp40) member C18 SH3RF1 SH3 domain containing 1.544156019 CRTC3 CREB regulated −3.217230716 ring finger 1 transcription coactivator 3 RGS16 regulator of G-protein 1.541382294 TNKS tankyrase −3.217230716 signaling 16 NCAPH non-SMC condensin I 1.540788228 GRM6 glutamate metabotropic −3.224966365 complex subunit H receptor 6 USP14 ubiquitin specific 1.540333713 SPSB1 splA/ryanodine receptor −3.255500733 peptidase 14 domain and SOCS box containing 1 RFT1 RFT1 homolog 1.54031759 PARP8 poly(ADP-ribose) −3.263034406 polymerase family member 8 SLC31A1 solute carrier family 31 1.540275536 KCNRG potassium channel regulator −3.263034406 member 1 TCTEX1D2 Tctex1 domain containing 1.538332378 POU6F1 POU class 6 homeobox 1 −3.268517714 2 TTF2 transcription termination 1.537871953 REV3L REV3 like, DNA directed −3.270528942 factor 2 polymerase zeta catalytic subunit ZFP7 zinc finger protein 1.5360529 TCF7 transcription factor 7 (T-cell −3.272419178 7(Zfp7) specific, HMG-box) G6PD2 glucose-6-phosphate 1.5360529 NME4 NME/NM23 nucleoside −3.283551423 dehydrogenase 2(G6pd2) diphosphate kinase 4 DEFB14 defensin beta 14(Defb14) 1.5360529 PLAUR plasminogen activator, −3.285402219 urokinase receptor SLC18A3 solute carrier family 18 1.5360529 CD4 CD4 molecule −3.285402219 member A3 AHNAK2 AHNAK nucleoprotein 2 1.5360529 ZMYND11 zinc finger MYND-type −3.293186363 containing 11 HOXC12 homeobox C12 1.5360529 ARMCX5 armadillo repeat containing, −3.298404158 X-linked 5 CEACAM16 carcinoembryonic antigen 1.5360529 LPHN1 Description Not Found −3.300123725 related cell adhesion molecule 16 MOSPD3 motile sperm domain 1.5360529 PIK3IP1 phosphoinositide-3-kinase −3.307428525 containing 3 interacting protein 1 DCTN1 dynactin subunit 1 1.5360529 ERDR1 erythroid differentiation −3.317651188 regulator 1(Erdr1) MYB MYB proto-oncogene, 1.5360529 PLD4 phospholipase D family −3.328444792 transcription factor member 4 GLIPR1L2 GLI pathogenesis-related 1.5360529 BMF Bcl2 modifying factor −3.336283388 1 like 2 ALDH1A3 aldehyde dehydrogenase 1.5360529 GALNT11 polypeptide N- −3.345118795 1 family member A3 acetylgalactosaminyl- transferase 11 SLC2A8 solute carrier family 2 1.5360529 LCN2 lipocalin 2 −3.378511623 member 8 SRC SRC proto-oncogene, 1.5360529 PAG1 phosphoprotein membrane −3.385431037 non-receptor tyrosine anchor with kinase glycosphingolipid microdomains 1 ZCCHC17 zinc finger CCHC-type 1.535618518 DTX1 deltex E3 ubiquitin ligase 1 −3.425576064 containing 17 HNRNPUL1 heterogeneous nuclear 1.534420207 RFFL ring finger and FYVE-like −3.426684082 ribonucleoprotein U like 1 domain containing E3 ubiquitin protein ligase TRIM68 tripartite motif containing 1.533057052 MAFF MAF bZIP transcription −3.429615964 68 factor F TPST1 tyrosylprotein 1.53140111 TOR1AIP2 torsin 1A interacting protein −3.432316325 sulfotransferase 1 2 OLFR922 olfactory receptor 1.531260941 SNN stannin −3.432316325 922(Olfr922) FIG4 FIG4 phosphoinositide 5- 1.530442167 CLEC4N C-type lectin domain family −3.433567144 phosphatase 4, member n(Clec4n) SETMAR SET domain and mariner 1.530442167 RREB1 ras responsive element −3.443780274 transposase fusion gene binding protein 1 GSTM5 glutathione S-transferase 1.530053218 CCDC84 coiled-coil domain −3.445188687 mu 5 containing 84 TUBA3B tubulin, alpha 1.527986221 ID3 inhibitor of DNA binding 3, −3.46350285 3B(Tuba3b) HLH protein PDCL phosducin like 1.527807072 BC065397 cDNA sequence −3.465974465 BC065397(BC065397) SMPDL3B sphingomyelin 1.527243888 VRK1 vaccinia related kinase 1 −3.46760555 phosphodiesterase acid like 3B ABHD14A abhydrolase domain 1.527213882 HOXD13 homeobox D13 −3.491853096 containing 14A TIPIN TIMELESS interacting 1.526972991 MAPK8IP2 mitogen-activated protein −3.491853096 protein kinase 8 interacting protein 2 DSCC1 DNA replication and 1.525986429 HOXA5 homeobox A5 −3.517275693 sister chromatid cohesion 1 PSMD1 proteasome 26S subunit, 1.525574957 HIST1H1A histone cluster 1, Hla −3.523561956 non-ATPase 1 BZRAP1 benzodiazepine receptor 1.524166255 MAML1 mastermind like −3.523603553 associated protein transcriptional coactivator 1 1(Bzrap1) ENO3 enolase 3 1.523778831 PTPDC1 protein tyrosine −3.526694846 phosphatase domain containing 1 E330034G19RIK RIKEN cDNA 1.523561956 TNFRSF12A tumor necrosis factor −3.528725998 E330034G19 receptor superfamily gene(E330034G19Rik) member 12A GABRP gamma-aminobutyric acid 1.523561956 TNIP2 TNFAIP3 interacting −3.539158811 type A receptor pi subunit protein 2 SLC14A2 solute carrier family 14 1.523561956 HIST2H4 histone cluster 2, −3.540773411 member 2 H4(Hist2h4) YWHAE tyrosine 3- 1.522478712 PIM2 Pim-2 proto-oncogene, −3.557655155 monooxygenase/tryptoph serine/threonine kinase an 5-monooxygenase activation protein epsilon EHBP1L1 EH domain binding 1.522282169 DOK7 docking protein 7 −3.567781854 protein 1 like 1 CHGB chromogranin B 1.51924262 TNFSF14 tumor necrosis factor −3.588895735 superfamily member 14 TXNRD2 thioredoxin reductase 2 1.519008256 TDRKH tudor and KH domain −3.590961241 containing NCF1 neutrophil cytosolic factor 1.518873761 FIBCD1 fibrinogen C domain −3.608656121 1 containing 1 OAF out at first homolog 1.517431856 RBBP9 RB binding protein 9, serine −3.608809243 hydrolase FAM110A family with sequence 1.517263583 DERL1 derlin 1 −3.617651119 similarity 110 member A ANGEL1 angel homolog 1 1.515832566 LENG9 leukocyte receptor cluster −3.62058641 (Drosophila) member 9 RTN4IP1 reticulon 4 interacting 1.515760776 TRPC2 transient receptor potential −3.62058641 protein 1 cation channel subfamily C member 2, pseudogene LAMP2 lysosomal associated 1.515709038 CCDC134 coiled-coil domain −3.632268216 membrane protein 2 containing 134 KRT4 keratin 4 1.514299789 OAS2 2′-5′-oligoadenylate −3.632268216 synthetase 2 PAFAH1B3 platelet activating factor 1.5142935 2410127L17RIK Description Not Found −3.646738698 acetylhydrolase 1b catalytic subunit 3 STT3A STT3A, catalytic subunit 1.513537695 RSAD1 radical S-adenosyl −3.649220471 of the methionine domain oligosaccharyltransferase containing 1 complex PRKAR1B protein kinase cAMP- 1.51340003 H2-DMB1 histocompatibility 2, class −3.649615459 dependent type I II, locus Mb1(H2-DMb1) regulatory subunit beta HIST1H2BB histone cluster 1, H2bb 1.512941595 IFT81 intraflagellar transport 81 −3.673839056 ZFP39 zinc finger protein 1.511385424 MID1 midline 1 −3.683696454 39(Zfp39) PLK1 polo like kinase 1 1.511151166 DEPDC1B DEP domain containing 1B −3.683696454 1700028P14RIK Description Not Found 1.510961919 SMAD3 SMAD family member 3 −3.716296166 D10BWG1379E Description Not Found 1.510961919 UBTD1 ubiquitin domain containing −3.716990894 1 TREM3 triggering receptor 1.510961919 FBXO44 F-box protein 44 −3.738767837 expressed on myeloid cells 3(Trem3) GM128 predicted gene 1.510961919 KCNMB4 potassium calcium- −3.741951111 128(Gm128) activated channel subfamily M regulatory beta subunit 4 OLFR741 olfactory receptor 1.510961919 FAIM3 Description Not Found −3.754887502 741(Olfr741) OLFR523 olfactory receptor 1.510961919 CCM2 CCM2 scaffolding protein −3.754887502 523(Olfr523) DCPP1 demilune cell and parotid 1.510961919 DAG1 dystroglycan 1 −3.760220946 protein 1(Dcpp1) RPRML reprimo like 1.510961919 FCGR3 Fc receptor, IgG, low −3.776103988 affinity III(Fcgr3) CHRD chordin 1.510961919 ZNRF1 zinc and ring finger 1, E3 −3.776103988 ubiquitin protein ligase C5AR1 complement component 1.510961919 TLR1 toll like receptor 1 −3.786596362 5a receptor 1 APOA2 apolipoprotein A2 1.510961919 HSD17B11 hydroxysteroid 17-beta −3.789207575 dehydrogenase 11 PRG2 proteoglycan 2, pro 1.510961919 ZPBP zona pellucida binding −3.887525271 eosinophil major basic protein protein VCAM1 vascular cell adhesion 1.510961919 ZSWIM3 zinc finger SWIM-type −3.892391026 molecule 1 containing 3 LY6G5B lymphocyte antigen 6 1.510961919 SOCS1 suppressor of cytokine −3.892391026 complex, locus G5B signaling 1 AIM2 absent in melanoma 2 1.510961919 KLF9 Kruppel like factor 9 −3.902021342 DMBX1 diencephalon/ 1.510961919 AHSA2 AHA1, activator of heat −3.904760449 mesencephalon shock 90 kDa protein homeobox 1 ATPase homolog 2 (yeast) HCN2 hyperpolarization 1.510961919 DDHD1 DDHD domain containing 1 −3.914086097 activated cyclic nucleotide gated potassium channel 2 MRGPRF MAS related GPR family 1.510961919 CNKSR3 CNKSR family member 3 −3.930737338 member F CYTH4 cytohesin 4 1.510961919 CPEB2 cytoplasmic −4.017516295 polyadenylation element binding protein 2 ANGPTL3 angiopoietin like 3 1.510961919 TRP53BP2 transformation related −4.021932279 protein 53 binding protein 2(Trp53bp2) DHX29 DEAH-box helicase 29 1.510667738 FAM178A family with sequence −4.03562391 similarity 178, member A(Fam178a) PMPCB peptidase, mitochondrial 1.509477625 RCN3 reticulocalbin 3 −4.03562391 processing beta subunit HRH3 histamine receptor H3 1.508554002 SPTLC2 serine palmitoyltransferase −4.040015679 long chain base subunit 2 ZFP282 zinc finger protein 1.507419453 ZFP810 zinc finger protein −4.070389328 282(Zfp282) 810(Zfp810) TBC1D7 TBC1 domain family 1.504847821 NAGA alpha-N- −4.074676686 member 7 acetylgalactosaminidase ARSB arylsulfatase B 1.504845728 KLRA20 killer cell lectin-like −4.078951341 receptor subfamily A, member 20(Klra20) RAD17 RAD17 checkpoint clamp 1.504177542 STK11IP serine/threonine kinase 11 −4.083213368 loader component interacting protein CMTM7 CKLF like MARVEL 1.503297831 KLF4 Kruppel like factor 4 −4.084306687 transmembrane domain containing 7 NFKB2 nuclear factor kappa B 1.500363085 INADL Description Not Found −4.086667018 subunit 2 TOP3A topoisomerase (DNA) III −1.50007357 URM1 ubiquitin related modifier 1 −4.0907078 alpha RAB33B RAB33B, member RAS −1.50054042 PELI1 pellino E3 ubiquitin protein −4.093813673 oncogene family ligase 1 LYSMD1 LysM domain containing −1.500614885 FBLN1 fibulin 1 −4.098032083 1 POLG2 polymerase (DNA) −1.500707646 HR hair growth associated −4.135452784 gamma 2, accessory subunit TGIF1 TGFB induced factor −1.501196523 ASB6 ankyrin repeat and SOCS −4.137503524 homeobox 1 box containing 6 RELL1 RELT like 1 −1.50300255 SLC27A5 solute carrier family 27 −4.141596278 member 5 CYP26B1 cytochrome P450 family −1.50439813 PPP1R3F protein phosphatase 1 −4.14974712 26 subfamily B member 1 regulatory subunit 3F PTRH2 peptidyl-tRNA hydrolase −1.504678598 AB124611 cDNA sequence −4.173373402 2 AB124611(AB124611) ZKSCAN3 zinc finger with KRAB −1.504916722 CD40 CD40 molecule −4.181897643 and SCAN domains 3 SP8 Sp8 transcription factor −1.505999092 SMAD5 SMAD family member 5 −4.183883459 SAMD14 sterile alpha motif domain −1.506272343 COL23A1 collagen type XXIII alpha 1 −4.221103725 containing 14 chain MX2 MX dynamin like GTPase −1.507268463 ZFP595 zinc finger protein −4.228818691 2 595(Zfp595) OCRL OCRL, inositol −1.507638755 PECAM1 platelet and endothelial cell −4.232789973 polyphosphate-5- adhesion molecule 1 phosphatase SYNJ2BP synaptojanin 2 binding −1.507669173 TMEM138 transmembrane protein 138 −4.241228289 protein CPLX4 complexin 4 −1.508554002 RFX2 regulatory factor X2 −4.244125943 LGALS9 galectin 9 −1.509246723 KCTD12 potassium channel −4.247846204 tetramerization domain containing 12 TAZ tafazzin −1.509269953 TRIM56 tripartite motif containing −4.262008929 56 2310002L09RIK Description Not Found −1.510961919 EIF4EBP2 eukaryotic translation −4.263034406 initiation factor 4E binding protein 2 ZFP97 zinc finger protein −1.510961919 RALGPS2 Ral GEF with PH domain −4.279842694 97(Zfp97) and SH3 binding motif 2 OLFR1494 olfactory receptor −1.510961919 TGM2 transglutaminase 2 −4.293161941 1494(Olfr1494) BC030867 cDNA sequence −1.510961919 ENC1 ectodermal-neural cortex 1 −4.311067102 BC030867(BC030867) CEACAM9 carcinoembryonic −1.510961919 LRIG1 leucine rich repeats and −4.375039431 antigen-related cell immunoglobulin like adhesion molecule domains 1 9(Ceacam9) LRIT1 leucine rich repeat, Ig-like −1.510961919 PRM1 protamine 1 −4.375039431 and transmembrane domains 1 KLK5 kallikrein related −1.510961919 DUSP7 dual specificity phosphatase −4.383538076 peptidase 5 7 KRT27 keratin 27 −1.510961919 SERTAD3 SERTA domain containing −4.399171094 3 CACNG4 calcium voltage-gated −1.510961919 KCNC1 potassium voltage-gated −4.409390936 channel auxiliary subunit channel subfamily C gamma 4 member 1 IL13RA1 interleukin 13 receptor −1.510961919 UBE2D3 ubiquitin conjugating −4.462706751 subunit alpha 1 enzyme E2 D3 TMEM121 transmembrane protein −1.510961919 SEPP1 selenoprotein P, plasma, 1 −4.463383458 121 HIST1H2AA histone cluster 1, H2aa −1.510961919 ADRB2 adrenoceptor beta 2 −4.463910999 MPZL3 myelin protein zero like 3 −1.510961919 PPP1R13B protein phosphatase 1 −4.471417658 regulatory subunit 13B TGFB2 transforming growth −1.510961919 ARRDC3 arrestin domain containing −4.504620392 factor beta 2 3 IFT74 intraflagellar transport 74 −1.510961919 GNGT2 G protein subunit gamma −4.531381461 transducin 2 FCRL1 Fc receptor like 1 −1.510961919 SIAH1A seven in absentia −4.539158811 1A(Siah1a) ADRB1 adrenoceptor beta 1 −1.510961919 XPC XPC complex subunit, −4.563768278 DNA damage recognition and repair factor MAGI2 membrane associated −1.510961919 HIPK1 homeodomain interacting −4.683696454 guanylate kinase, WW protein kinase 1 and PDZ domain containing 2 SCG5 secretogranin V −1.510961919 H2-OB histocompatibility 2, O −4.700439718 region beta locus(H2-Ob) GCK glucokinase −1.510961919 BACH2 BTB domain and CNC −4.716990894 homolog 2 ASB10 ankyrin repeat and SOCS −1.510961919 MAPILC3A microtubule associated −4.722466024 box containing 10 protein 1 light chain 3 alpha SELE selectin E −1.510961919 LRRFIP1 LRR binding FLII −4.761551232 interacting protein 1 IGFBP3 insulin like growth factor −1.510961919 ATP10D ATPase phospholipid −4.766581958 binding protein 3 transporting 10D (putative) TPT1 tumor protein, −1.510961919 IGFBP4 insulin like growth factor −4.790993785 translationally-controlled binding protein 4 1 ROCK1 Rho associated coiled-coil −1.510961919 TMEM108 transmembrane protein 108 −4.865423978 containing protein kinase 1 OGFRL1 opioid growth factor −1.510961919 PTK2 protein tyrosine kinase 2 −4.875719796 receptor-like 1 TMEM38A transmembrane protein −1.510961919 CLEC11A C-type lectin domain family −4.897240426 38A 11 member A RLTPR Description Not Found −1.51227339 LRP12 LDL receptor related −4.955029571 protein 12 ITPKC inositol-trisphosphate 3- −1.512389725 GCNT2 glucosaminyl (N-acetyl) −4.958842675 kinase C transferase 2, I-branching enzyme (I blood group) TLE4 transducin like enhancer −1.51341989 F10 coagulation factor X −4.965784285 of split 4 PDE4D phosphodiesterase 4D −1.513667908 DBP D-box binding PAR bZIP −4.966549451 transcription factor A130010J15RIK Description Not Found −1.514296211 ABCG1 ATP binding cassette −5.002252452 subfamily G member 1 RNF167 ring finger protein 167 −1.514765492 WDR78 WD repeat domain 78 −5.017921908 CCBL1 Description Not Found −1.515626494 DNAJC6 DnaJ heat shock protein −5.017921908 family (Hsp40) member C6 HSD17B1 hydroxysteroid 17-beta −1.516875069 AFF4 AF4/FMR2 family member −5.033423002 dehydrogenase 1 4 OSM oncostatin M −1.517234668 TNFRSF26 tumor necrosis factor −5.040015679 receptor superfamily, member 26(Tnfrsf26) RHPN1 rhophilin, Rho GTPase −1.517275693 GFOD2 glucose-fructose −5.070389328 binding protein 1 oxidoreductase domain containing 2 TAS2R105 taste receptor, type 2, −1.517431856 TYROBP TYRO protein tyrosine −5.114783447 member 105(Tas2r105) kinase binding protein NIPBL NIPBL, cohesin loading −1.517569618 TMEM176B transmembrane protein −5.118941073 factor 176B CXCR3 C-X-C motif chemokine −1.519325267 ZFP710 zinc finger protein −5.159871337 receptor 3 710(Zfp710) SMURF1 SMAD specific E3 −1.520263252 ENPP4 ectonucleotide −5.181897643 ubiquitin protein ligase 1 pyrophosphatase/ phosphodiesterase 4 (putative) RNF208 ring finger protein 208 −1.52126647 MAPK8 mitogen-activated protein −5.259272487 kinase 8 ITGA5 integrin subunit alpha 5 −1.523517983 TNFRSF25 tumor necrosis factor −5.289096702 receptor superfamily member 25 USP18 ubiquitin specific −1.524814077 LCN4 lipocalin 4(Lcn4) −5.366322214 peptidase 18 PIP5K1A phosphatidylinositol-4- −1.525074369 CRIM1 cysteine rich −5.369815424 phosphate 5-kinase type 1 transmembrane BMP alpha regulator 1 STRBP spermatid perinuclear −1.52561213 RTP4 receptor transporter protein −5.444600814 RNA binding protein 4 GRAMD2 GRAM domain −1.52652805 PRNP prion protein −5.495055528 containing 2 ZFP101 zinc finger protein −1.526555668 ZFP747 zinc finger protein −5.496654083 101(Zfp101) 747(Zfp747) RUNDC1 RUN domain containing 1 −1.526563287 CD7 CD7 molecule −5.504620392 SLC13A3 solute carrier family 13 −1.528487927 ARHGAP26 Rho GTPase activating −5.548436625 member 3 protein 26 CCDC94 coiled-coil domain −1.528487927 S100A9 S100 calcium binding −5.557655155 containing 94 protein A9 MRPS14 mitochondrial ribosomal −1.528962318 AQP9 aquaporin 9 −5.572889668 protein S14 NEU4 neuraminidase 4 −1.529820947 CXCR5 C-X-C motif chemokine −5.573647187 (sialidase) receptor 5 PCGF1 polycomb group ring −1.53059536 CCNO cyclin O −5.574404309 finger 1 PNPLA7 patatin like phospholipase −1.53207883 LYNX1 Ly6/neurotoxin 1 −5.666756592 domain containing 7 SPATA19 spermatogenesis −1.533014103 CLDN10 claudin 10 −5.782015335 associated 19 AP4B1 adaptor related protein −1.533821865 AMIGO2 adhesion molecule with Ig- −5.83541884 complex 4 beta 1 subunit like domain 2 BC068281 cDNA sequence −1.5360529 CD79B CD79b molecule −5.94016675 BC068281(BC068281) GK2 glycerol kinase 2 −1.5360529 USP53 ubiquitin specific peptidase −5.980710829 53 PIGM phosphatidylinositol −1.5360529 IKBKE inhibitor of kappa light −6.005624549 glycan anchor polypeptide gene enhancer biosynthesis class M in B-cells, kinase epsilon FKBP6 FK506 binding protein 6 −1.5360529 ALOX5AP arachidonate 5- −6.008988783 lipoxygenase activating protein EVI5 ecotropic viral integration −1.5360529 GGT1 gamma-glutamyltransferase −6.010108453 site 5 1 BCL11A B-cell CLL/lymphoma −1.5360529 CAMK2D calcium/calmodulin −6.047669251 11A dependent protein kinase II delta PER1 period circadian clock 1 −1.537278499 RAB3D RAB3D, member RAS −6.156841525 oncogene family BTBD9 BTB domain containing 9 −1.537451456 MAP3K8 mitogen-activated protein −6.376776572 kinase kinase kinase 8 USP38 ubiquitin specific −1.537763627 NOTCH4 notch 4 −6.495055528 peptidase 38 LRRC57 leucine rich repeat −1.538083341 MACROD1 MACRO domain −6.581200582 containing 57 containing 1 5830415F09RIK Description Not Found −1.53855912 RNF144A ring finger protein 144A −6.632268216 EGR2 early growth response 2 −1.540038325 PDE2A phosphodiesterase 2A −6.86913112 GMEB2 glucocorticoid −1.541122795 THA1 threonine aldolase 1(Tha1) −6.885086225 modulatory element binding protein 2 PIK3R4 phosphoinositide-3-kinase −1.541975323 APP amyloid beta precursor −6.940754047 regulatory subunit 4 protein KRR1 KRR1, small subunit −1.54225805 FAM109A family with sequence −6.968666793 processome component similarity 109 member A homolog COL9A1 collagen type IX alpha 1 −1.54225805 LRG1 leucine rich alpha-2- −6.995484519 glycoprotein 1 POLD4 polymerase (DNA) delta −1.542654605 IL11RA1 interleukin 11 receptor, −7.016251155 4, accessory subunit alpha chain 1(Il11ra1) ACSS2 acyl-CoA synthetase −1.544045378 CNR2 cannabinoid receptor 2 −7.213347282 short-chain family member 2 PDLIM1 PDZ and LIM domain 1 −1.544785186 NUAK2 NUAK family kinase 2 −7.369815424 A430107P09RIK Description Not Found −1.544921568 GPR146 G protein-coupled receptor −7.577806447 146 SLC38A11 solute carrier family 38 −1.546222547 member 11 *Log2Fold Change = log2(4 + L+/4 − L−)

To investigate the molecular pathways between these three populations, gene ontology networks were grouped into nodes and the most significant pathways within each node were determined (FIG. 6A). Gene ontology (GO) terms shared between our dysfunctional T cell dataset and the published hypofunctional T cell dataset were greatly enriched in cell cycle genes, consistent with the observation that the dysfunctional population is largely Ki67⁺. GO terms shared between dysfunctional and exhausted gene sets encompassed effector programs such as regulation of cell killing, chemotaxis, interferon-7 production. GO terms shared between hypofunctional and exhausted gene sets consisted of cell cycle pathways, negative regulation of lymphocytes, and interferon-7 production. These data indicate that while some conserved molecular programs likely exist in these dysfunctional differentiation states, many pathways may be differentially regulated between chronic viral infections and in the tumor context. While many inhibitory receptors, including Pdcd1 (PD-1), Havcr2 (TIM-3), Cd244 (2B4), Klre1, and Lag3 were shared between all data sets; the co-stimulatory receptors Tnfrsf4 (OX-40) and Tnfrsf9 (4-1BB) were upregulated in dysfunctional and hypofunctional CD8⁺ TIL data sets. Therefore, to enrich in potential markers and therapeutic targets on tumor specific CD8⁺ TILs, the complete cell surface phenotype of the 4-1BB⁺LAG-3⁺CD8 TIL population was characterized. Comparing the different CD8⁺ TIL subpopulations, several additional upregulated co-stimulatory receptors were found: Tnfrsf18 (GITR), Nkg2d (KLRK1) and Cd27. The transcript for Nrp1 (neuropilin-1), which encodes for a cell surface receptor protein implicated in CD4⁺ Treg function (Sarris et al., 2008; incorporated by reference in its entirety), was also highly expressed. Expression of many of these molecules was confirmed by flow cytometry at day 7, 14 and 21 after tumor inoculation (FIG. 6C). The analysis was extended to include the co-stimulatory molecules ICOS and CD160 and the inhibitory receptor T cell immunoreceptor with Ig and ITIM domains (TIGIT) because ICOS and CD160 were close to the cutoff value and no probe was present for TIGIT in the gene array. In addition, recent reports indicate that targeting these receptors can be therapeutic in murine models of cancer (Johnston et al., 2014; Fan et al., 2014; incorporated by reference in their entireties). PD-1, TIGIT, TIM-3, CD27 and NRP1 were expressed the majority of the 4-1BB⁺LAG-3⁺ TIL population and expression was maintained over time. 2B4, CD160, CTLA4, OX-40, and GITR subdivided a lesser fraction of the 4-1BB⁺LAG-3⁺ population. The expression of several inhibitory receptors, 2B4, TIM3 and CD160 increased over this 3-week time frame while expression of the co-stimulatory receptors, ICOS and OX-40, decreased (FIG. 6C).

To address if the dysfunctional CD8⁺ TILs are terminally-differentiated short term effector cells or memory-like cells, the expression of KLRG-1 and IL-7Rα (Joshi et al., 2007). Most of the CD8⁺ TIL were negative for KLRG-1 expression and there was no difference between the 4-1BB⁺LAG-3⁺ and 4-1BB⁻LAG-3 populations. However, the majority of the 4-1BB⁺LAG-3⁺ TIL did not express the IL-7 receptor (IL-7Ra) compared to their negative counter parts (FIG. 6D). These results indicate that the 4-1BB⁻LAG-3 TIL, which are not apparently specific for antigens expressed in the tumor microenvironment, are more memory-like, yet at the same time the tumor antigen-specific LAG-3⁺4-1BB⁺ subset has not fully acquired a terminal effector phenotype.

Functional Relevance of Genes that are Differentially Regulated in CD8⁺4-1BB⁺LAG-3⁺ TILs

The gene array results in Table 2 provide a list of genes characterizing CD8⁺4-1BB⁺LAG-3⁺ TILs. The list includes therapeutic targets and additional markers of anti-tumor immunity. Experiments conducted during development of embodiments herein to test the functional relevance of these additional targets/markers (FIG. 11 ). Data indicate that the array has identified targets for immunotherapy, using knockout mice (e.g., PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.). Experiments demonstrate that Nrn1 and CRTAM are negative regulators of the anti-tumor immune response, as knockout mice lacking either of these molecules showed improved immune-mediated tumor control in vivo. In contrast, Sema7a is a positive regulator of anti-tumor immune responses, as knockout mice lacking this molecule show diminished immune-mediated tumor control in vivo (FIG. 11 ). These experiments indicate that agonists of Sema7a signaling and antagonists of Nrn1 and/or CRTAM should be useful therapeutics for the treatment of cancer.

Targeting 4-1BB and LAG-3 Exerts Anti-Tumor Activity In Vivo and Normalizes the Function and Phenotypic Composition of CD8⁺ TILs

Experiments were conducted during development of embodiments herein to assess whether targeting these receptors might have therapeutic utility. To this end, an agonistic anti-4-1BB mAb was administered alone or in combination with a blocking anti-LAG-3 mAb in mice bearing established B16. SIY tumors. While each antibody treatment alone had some therapeutic effect as reflected by slower tumor growth, the combination was particularly potent (FIG. 7A). Analysis of the tumor microenvironment revealed that improved tumor control with the combination therapy was accompanied by an increase in the number of CD8⁺ TILs specific for the SIY antigen (FIG. 7B), consistent with results reported previously with anti-PD-L1⁺ anti-CTLA-4 mAb (Spranger et al., 2014b; Twyman-Saint Victor et al., 2015; incorporated by reference in their entireties).

It was next examined whether the therapeutic effect of anti-4-1BB+anti-LAG-3 mAbs was associated with a loss of phenotypic markers defining dysfunctional T cells in the steady state. Due to concern that re-analyzing the T cells for expression of LAG-3 and 4-1BB might be problematic, as the administered Abs could theoretically modulate the target receptors from the cell surface, the coordinate expression of additional receptors as identified above by gene expression profiling was taken advantage of. Preliminary analyses of the bulk TIL subpopulations revealed decreased expression of NRP1 and 2B4 following anti-LAG-3+anti-4-1BB treatment (data not shown). Co-expression of 2B4 and NRP1 on SIY-reactive CD8⁺ TILs identified by pentamer staining was analyzed. A 2.7-fold-decrease in the co-expression of 2B4 and NRP1 was observed upon anti-4-1BB+ and anti-LAG-3 mAb treatment (FIG. 7C), indicating a loss of the surface phenotype associated with T cell dysfunction. To determine whether this change was accompanied by a shift towards an effector phenotype, expression of KLGR-1 was examined. Indeed, a marked increase in KLGR-1 expression was observed on the SIY-reactive TIL following treatment, and a 3.7-fold increase in the KLRG-1^(hi)IL-7RA^(lo) population was observed (FIG. 7D).

To eliminate the possibility that treatment with anti-LAG-3+anti-4-1BB mAbs was not altering the phenotype of T cells already within the tumor but rather was supporting recruitment of newly primed functional T cells from secondary lymphoid organs, the S1PR inhibitor FTY720, which prevents T cell egress from lymph nodes (Halin et al., 2005; incorporated by reference in its entirety), was utilized. We The efficacy of anti-PD-L1-based immunotherapies was preserved in the presence of FTY720, arguing for re-functionalization of TIL as the major mechanism of action (Spranger et al., 2014a; incorporated by reference in its entirety). FTY720 administration was started on day 6 after tumor inoculation, 24 hours before the start of anti-LAG-3+anti-4-1BB treatment, and continued every day until TIL analysis on day 14. Peripheral blood analyzed at the same time point revealed marked depletion of circulating T cells (FIG. 9 ). Despite this loss of circulating T cells, the down regulation of 2B4 and NRP1 and the shift towards the KLRG1^(hi)IL-7RA^(lo) phenotype was nonetheless preserved (FIGS. 7E and F). To examine functional restoration of the TIL, the KLRG-1^(lo)IL-7RA^(lo) and KLRG-1^(hi)IL-7RA^(lo)CD8⁺ TIL populations were sorted from B16. SIY tumors on day 14 following treatment and analyzed for IL-2 after restimulation in vitro. Indeed, the KLRG-1^(lo)IL-7RA^(lo) and KLRG-1^(hi)IL-7RA^(lo) populations showed an increased capacity to produce IL-2 upon stimulation (FIG. 7G). The relative level of Il-2 mRNA was comparable between the two CD8⁺ TIL populations and control CD8⁺CD44⁺ TdLN T cells. Collectively, these data indicate that anti-4-1BB/anti-LAG-3 combinatorial treatment induces significant changes in the phenotype profile and promotes functional restoration of tumor antigen-specific CD8⁺ T cells already present within the tumor microenvironment.

REFERENCES

The following references, some of which are cited above, are herein incorporated by references in their entireties.

-   Ahmadzadeh, M., L. A. Johnson, B. Heemskerk, J. R. Wunderlich, M. E.     Dudley, D. E. White, and S. A. Rosenberg. 2009. Tumor     antigen-specific CD8 T cells infiltrating the tumor express high     levels of PD-1 and are functionally impaired. Blood. 114:1537-1544.     doi:10.1182/blood-2008-12-195792. -   Baitsch, L., A. Legat, L. Barba, S. A. Fuertes Marraco, J.-P.     Rivals, P. Baumgaertner, C. Christiansen-Jucht, H. Bouzourene, D.     Rimoldi, H. Pircher, N. Rufer, M. Matter, O. Michielin, and D. E.     Speiser. 2012. Extended co-expression of inhibitory receptors by     human CD8 T-cells depending on differentiation, antigen-specificity     and anatomical localization. PLoS ONE. 7:e30852.     doi:10.1371/journal.pone.0030852. -   Baitsch, L., P. Baumgaertner, E. Devêvre, S. K. Raghav, A. Legat, L.     Barba, S. Wieckowski, H. Bouzourene, B. Deplancke, P. Romero, N.     Rufer, and D. E. Speiser. 2011. Exhaustion of tumor-specific CD8⁺ T     cells in metastases from melanoma patients. J. Clin. Invest.     121:2350-2360. doi:10.1172/JC146102. -   Blackburn, S. D., H. Shin, W. N. Haining, T. Zou, C. J. Workman, A.     Polley, M. R. Betts, G. J. Freeman, D. A. A. Vignali, and E. J.     Wherry. 2009. Coregulation of CD8+ T cell exhaustion by multiple     inhibitory receptors during chronic viral infection. Nature     Publishing Group. 10:29-37. doi:10.1038/ni.1679. -   Brown, I. E., C. Blank, J. Kline, A. K. Kacha, and T. F.     Gajewski. 2006. Homeostatic proliferation as an isolated variable     reverses CD8+ T cell anergy and promotes tumor rejection. Journal of     Immunology (Baltimore, Md.: 1950). 177:4521-4529. -   Clouthier, D. L., A. C. Zhou, and T. H. Watts. 2014. Anti-GITR     agonist therapy intrinsically enhances CD8 T cell responses to     chronic lymphocytic choriomeningitis virus (LCMV), thereby     circumventing LCMV-induced downregulation of costimulatory GITR     ligand on APC. The Journal of Immunology. 193:5033-5043.     doi:10.4049/jimmunol.1401002. -   Crawford, A., J. M. Angelosanto, C. Kao, T. A. Doering, P. M.     Odorizzi, B. E. Barnett, and E. J. Wherry. 2014. Molecular and     Transcriptional Basis of CD4(+) T Cell Dysfunction during Chronic     Infection. Immunity. 40:289-302. doi:10.1016/j.immuni.2014.01.005. -   Cunningham, C. R., A. Champhekar, M. V. Tullius, B. J. Dillon, A.     Zhen, J. R. de la Fuente, J. Herskovitz, H. Elsaesser, L. M.     Snell, E. B. Wilson, J. C. de la Torre, S. G. Kitchen, M. A.     Horwitz, S. J. Bensinger, S. T. Smale, and D. G. Brooks. 2016. Type     I and Type II Interferon Coordinately Regulate Suppressive Dendritic     Cell Fate and Function during Viral Persistence. PLoS Pathog.     12:e1005356. doi:10.1371/journal.ppat.1005356. -   Currier, J. R., and M. A. Robinson. 2001. Spectratype/Immunoscope     Analysis of the Expressed TCR Repertoire. John Wiley & Sons, Inc.,     Hoboken, NJ, USA. 92544 pp. -   Diamond, M. S., M. Kinder, H. Matsushita, M. Mashayekhi, G. P.     Dunn, J. M. Archambault, H. Lee, C. D. Arthur, J. M. White, U.     Kalinke, K. M. Murphy, and R. D. Schreiber. 2011. Type I interferon     is selectively required by dendritic cells for immune rejection of     tumors. J. Exp. Med. 208:1989-2003. doi:10.1084/jem.20101158. -   Doering, T. A., A. Crawford, J. M. Angelosanto, M. A. Paley, C. G.     Ziegler, and E. J. Wherry. 2012. Network analysis reveals centrally     connected genes and pathways involved in CD8+ T cell exhaustion     versus memory. Immunity. 37:1130-1144.     doi:10.1016/j.immuni.2012.08.021. -   Evaristo, C., S. Spranger, S. E. Barnes, M. L. Miller, L. L.     Molinero, F. L. Locke, T. F. Gajewski, and M.-L. Alegre. 2016.     Cutting Edge: Engineering Active IKKβ in T Cells Drives Tumor     Rejection. The Journal of Immunology. 196:2933-2938.     doi:10.4049/jimmunol.1501144. -   Fan, X., S. A. Quezada, M. A. Sepulveda, P. Sharma, and J. P.     Allison. 2014. Engagement of the ICOS pathway markedly enhances     efficacy of CTLA-4 blockade in cancer immunotherapy. J. Exp. Med.     211:715-725. doi:10.1084/jem.20130590. -   Fourcade, J., Z. Sun, O. Pagliano, P. Guillaume, I. F. Luescher, C.     Sander, J. M. Kirkwood, D. Olive, V. Kuchroo, and H. M.     Zarour. 2012. CD8(+) T cells specific for tumor antigens can be     rendered dysfunctional by the tumor microenvironment through     upregulation of the inhibitory receptors BTLA and PD-1. Cancer Res.     72:887-896. doi:10.1158/0008-5472. CAN-11-2637. -   Fridman, W. H., F. Pagès, C. Sautès-Fridman, and J. Galon. 2012. The     immune contexture in human tumours: impact on clinical outcome.     Nature Publishing Group. 12:298-306. doi:10.1038/nrc3245. -   Fuertes, M. B., A. K. Kacha, J. Kline, S.-R. Woo, D. M. Kranz, K. M.     Murphy, and T. F. Gajewski. 2011. Host type I IFN signals are     required for antitumor CD8+ T cell responses through     CD8{alpha}+dendritic cells. J. Exp. Med. 208:2005-2016.     doi:10.1084/jem.20101159. -   Gajewski, T. F. 2007a. The expanding universe of regulatory T cell     subsets in cancer. Immunity. 27:185-187.     doi:10.1016/j.immuni.2007.08.001. -   Gajewski, T. F. 2007b. Failure at the Effector Phase: Immune     Barriers at the Level of the Melanoma Tumor Microenvironment. Clin.     Cancer Res. 13:5256-5261. doi:10.1158/1078-0432. CCR-07-0892. -   Gajewski, T. F., H. Schreiber, and Y.-X. Fu. 2013. Innate and     adaptive immune cells in the tumor microenvironment. Nat Immunol.     14:1014-1022. doi:10.1038/ni.2703. -   Gajewski, T. F., Y. Meng, C. Blank, I. E. Brown, A. K. Kacha, J.     Kline, and H. Harlin. 2006. Immune resistance orchestrated by the     tumor microenvironment. Immunological Reviews. 1-15. -   Goldszmid, R. S., A. Dzutsev, and G. Trinchieri. 2014. Host Immune     Response to Infection and Cancer: Unexpected Commonalities. Cell     Host and Microbe. 15:295-305. doi:10.1016/j.chom.2014.02.003. -   Gros, A., P. F. Robbins, X. Yao, Y. F. Li, S. Turcotte, E.     Tran, J. R. Wunderlich, A. Mixon, S. Farid, M. E. Dudley, K.-I.     Hanada, J. R. Almeida, S. Darko, D. C. Douek, J. C. Yang, and S. A.     Rosenberg. 2014. PD-1 identifies the patient-specific CD8⁺     tumor-reactive repertoire infiltrating human tumors. J. Clin.     Invest. 124:2246-2259. doi:10.1172/JCI73639. -   Halin, C., M. L. Scimone, R. Bonasio, J.-M. Gauguet, T. R.     Mempel, E. Quackenbush, R. L. Proia, S. Mandala, and U. H. von     Andrian. 2005. The SlP-analog FTY720 differentially modulates T-cell     homing via HEV: T-cell-expressed SlPl amplifies integrin activation     in peripheral lymph nodes but not in Peyer patches. Blood.     106:1314-1322. doi:10.1182/blood-2004-09-3687. -   Harlin, H., T. V. Kuna, A. C. Peterson, Y. Meng, and T. F.     Gajewski. 2006. Tumor progression despite massive influx of     activated CD8+ T cells in a patient with malignant melanoma ascites.     Cancer Immunol Immunother. 55:1185-1197.     doi:10.1007/s00262-005-0118-2. -   Harlin, H., Y. Meng, A. C. Peterson, Y. Zha, M. Tretiakova, C.     Slingluff, M. McKee, and T. F. Gajewski. 2009. Chemokine expression     in melanoma metastases associated with CD8+ T-cell recruitment.     Cancer Res. 69:3077-3085. doi:10.1158/0008-5472. CAN-08-2281. -   Hoelzinger, D. B., S. E. Smith, N. Mirza, A. L. Dominguez, S. Z.     Manrique, and J. Lustgarten. 2010. Blockade of CCL1 Inhibits T     Regulatory Cell Suppressive Function Enhancing Tumor Immunity     without Affecting T Effector Responses. The Journal of Immunology.     184:6833-6842. doi:10.4049/jimmunol.0904084. -   Jenkins, M. K., D. M. Pardoll, J. Mizuguchi, T. M. Chused, and R. H.     Schwartz. 1987. Molecular events in the induction of a nonresponsive     state in interleukin 2-producing helper T-lymphocyte clones. PNAS.     84:5409-5413. -   Johnston, R. J., L. Comps-Agrar, J. Hackney, X. Yu, M. Huseni, Y.     Yang, S. Park, V. Javinal, H. Chiu, B. Irving, D. L. Eaton,     and J. L. Grogan. 2014. The immunoreceptor TIGIT regulates antitumor     and antiviral CD8(+) T cell effector function. Cancer Cell.     26:923-937. doi:10.1016/j.ccell.2014.10.018. -   Joshi, N. S., W. Cui, A. Chandele, H. K. Lee, D. R. Urso, J.     Hagman, L. Gapin, and S. M. Kaech. 2007. Inflammation Directs Memory     Precursor and Short-Lived Effector CD8+ T Cell Fates via the Graded     Expression of T-bet Transcription Factor. Immunity. 27:281-295.     doi:10.1016/j.immuni.2007.07.010. -   Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh,     and R. Ahmed. 2003. Selective expression of the interleukin 7     receptor identifies effector CD8 T cells that give rise to     long-lived memory cells. Nat Immunol. 4:1191-1198.     doi:10.1038/ni1009. -   Kearse, M., R. Moir, A. Wilson, S. Stones-Havas, M. Cheung, S.     Sturrock, S. Buxton, A. Cooper, S. Markowitz, C. Duran, T.     Thierer, B. Ashton, P. Meintjes, and A. Drummond. 2012. Geneious     Basic: an integrated and extendable desktop software platform for     the organization and analysis of sequence data. Bioinformatics.     28:1647-1649. doi:10.1093/bioinformatics/bts199. -   Kim, J.-O., H. W. Kim, K.-M. Baek, and C.-Y. Kang. 2003. NF-κB and     AP-1 regulate activation-dependent CD137 (4-1BB) expression in T     cells. FEBS Letters. 541:163-170. doi:10.1016/50014-5793(03)00326-0. -   Kline, J., L. Zhang, L. Battaglia, K. S. Cohen, and T. F.     Gajewski. 2012. Cellular and molecular requirements for rejection of     B16 melanoma in the setting of regulatory T cell depletion and     homeostatic proliferation. The Journal of Immunology. 188:2630-2642.     doi:10.4049/jimmunol.1100845. -   Kuchroo, V. K., A. C. Anderson, and C. Petrovas. 2014. Coinhibitory     receptors and CD8 T cell exhaustion in chronic infections. Current     Opinion in HIV and AIDS. 9:439-445.     doi:10.1097/COH.0000000000000088. -   Larkin, J., F. S. Hodi, and J. D. Wolchok. 2015. Combined Nivolumab     and Ipilimumab or Monotherapy in Untreated Melanoma. New England     Journal of Medicine. 373:1270-1271. doi:10.1056/NEJMc1509660. -   Li, S., T. Miao, M. Sebastian, P. Bhullar, E. Ghaffari, M.     Liu, A. L. J. Symonds, and P. Wang. 2012. The transcription factors     Egr2 and Egr3 are essential for the control of inflammation and     antigen-induced proliferation of B and T cells. Immunity.     37:685-696. doi:10.1016/j.immuni.2012.08.001. -   Martinez, G. J., R. M. Pereira, T. Äijö, E. Y. Kim, F.     Marangoni, M. E. Pipkin, S. Togher, V. Heissmeyer, Y. C. Zhang, S.     Crotty, E. D. Lamperti, K. M. Ansel, T. R. Mempel, H.     Lähdesmäki, P. G. Hogan, and A. Rao. 2015. The Transcription Factor     NFAT Promotes Exhaustion of Activated CD8+ T Cells. Immunity.     42:265-278. doi:10.1016/j.immuni.2015.01.006. -   Odorizzi, P. M., K. E. Pauken, M. A. Paley, A. Sharpe, and E. J.     Wherry. 2015. Genetic absence of PD-1 promotes accumulation of     terminally differentiated exhausted CD8+ T cells. J. Exp. Med.     439:jem.20142237-1137. doi:10.1084/jem.20142237. -   Palazón, A., I. Martínez-Forero, A. Teijeira, A.     Morales-Kastresana, C. Alfaro, M. F. Sanmamed, J. L.     Perez-Gracia, I. Peñuelas, S. Hervás-Stubbs, A. Rouzaut, M. O. de     Landázuri, M. Jure-Kunkel, J. Aragonés, and I. Melero. 2012. The     HIF-1α hypoxia response in tumor-infiltrating T lymphocytes induces     functional CD137 (4-1BB) for immunotherapy. Cancer Discov.     2:608-623. doi:10.1158/2159-8290. CD-11-0314. -   Pardoll, D. M. 2012. The blockade of immune checkpoints in cancer     immunotherapy. 1-13. doi:10.1038/nrc3239. -   Pauken, K. E., and E. J. Wherry. 2015. Overcoming T cell exhaustion     in infection and cancer. Trends in Immunology. 36:265-276.     doi:10.1016/j.it.2015.02.008. -   Pearce, E. L., M. C. Poffenberger, C. H. Chang, and R. G.     Jones. 2013. Fueling immunity: insights into metabolism and     lymphocyte function. Science. -   Plaisier, S. B., R. Taschereau, J. A. Wong, and T. G. Graeber. 2010.     Rank-rank hypergeometric overlap: identification of statistically     significant overlap between gene-expression signatures. Nucl. Acids     Res. 38:e169-el69. doi:10.1093/nar/gkg636. -   Rosenblatt, J. D., and J. L. Stein. 2014. RRHO: Test overlap using     the Rank-Rank Hypergeometric test. -   Safford, M., S. Collins, M. A. Lutz, A. Allen, C.-T. Huang, J.     Kowalski, A. Blackford, M. R. Horton, C. Drake, R. H. Schwartz,     and J. D. Powell. 2005. Egr-2 and Egr-3 are negative regulators of T     cell activation. Nat Immunol. 6:472-480. doi:10.1038/ni1193. -   Sarkar, S., V. Kalia, W. N. Haining, B. T. Konieczny, S.     Subramaniam, and R. Ahmed. 2008. Functional and genomic profiling of     effector CD8 T cell subsets with distinct memory fates. J. Exp. Med.     205:625-640. doi:10.1084/jem.20071641. -   Sarris, M., K. G. Andersen, F. Randow, L. Mayr, and A. G.     Betz. 2008. Neuropilin-1 Expression on Regulatory T Cells Enhances     Their Interactions with Dendritic Cells during Antigen Recognition.     Immunity. 28:402-413. doi:10.1016/j.immuni.2008.01.012. -   Schietinger, A., and P. D. Greenberg. 2014. Tolerance and     exhaustion: defining mechanisms of T cell dysfunction. Trends in     Immunology. 35:51-60. doi:10.1016/j.it.2013.10.001. -   Schietinger, A., M. Philip, V. E. Krisnawan, E. Y. Chiu, J. J.     Delrow, R. S. Basom, P. Lauer, D. G. Brockstedt, S. E.     Knoblaugh, G. J. Hammerling, T. D. Schell, N. Garbi, and P. D.     Greenberg. 2016. Tumor-Specific T Cell Dysfunction Is a Dynamic     Antigen-Driven Differentiation Program Initiated Early during     Tumorigenesis. Immunity. doi:10.1016/j.immuni.2016.07.011. -   Schwartz, R. H. 2003. T C ELLA NERGY*. Annu. Rev. Immunol.     21:305-334. doi:10.1146/annurev.immunol.21.120601.141110. -   Schwartz, R. H., D. L. Mueller, M. K. Jenkins, and H. Quill. 1989.     T-cell clonal anergy. Cold Spring Harb. Symp. Quant. Biol. 54 Pt     2:605-610. -   Shannon, P., A. Markiel, O. Ozier, N. S. Baliga, J. T. Wang, D.     Ramage, N. Amin, B. Schwikowski, and T. Ideker. 2003. Cytoscape: a     software environment for integrated models of biomolecular     interaction networks. Genome Res. 13:2498-2504.     doi:10.1101/gr.1239303. -   Snell, L. M., and D. G. Brooks. 2015. New insights into type I     interferon and the immunopathogenesis of persistent viral     infections. Curr. Opin. Immunol. 34:91-98.     doi:10.1016/j.coi.2015.03.002. -   Spiotto, M. T., P. Yu, D. A. Rowley, M. I. Nishimura, S. C.     Meredith, T. F. Gajewski, Y.-X. Fu, and H. Schreiber. 2002.     Increasing Tumor Antigen Expression Overcomes “Ignorance” to Solid     Tumors via Crosspresentation by Bone Marrow-Derived Stromal Cells.     Immunity. 17:737-747. doi:10.1016/S1074-7613(02)00480-6. -   Spranger, S., H. K. Koblish, and B. Horton. 2014a. Mechanism of     tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade     involves restored IL-2 production and proliferation of CD8+ T cells     directly . . . Journal for . . . . -   Spranger, S., H. K. Koblish, B. Horton, P. A. Scherle, R. Newton,     and T. F. Gajewski. 2014b. Mechanism of tumor rejection with     doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored     IL-2 production and proliferation of CD8(+) T cells directly within     the tumor microenvironment. J Immunother Cancer. 2:3.     doi:10.1186/2051-1426-2-3. -   Spranger, S., R. M. Spaapen, Y. Zha, J. Williams, Y. Meng, T. T. Ha,     and T. F. Gajewski. 2013. Up-Regulation of PD-L1, IDO, and Tregs in     the Melanoma Tumor Microenvironment Is Driven by CD8+ T Cells.     Science Translational Medicine. 5:200ra116-200ra116.     doi:10.1126/scitranslmed.3006504. -   Sumitomo, S., K. Fujio, T. Okamura, and K. Yamamoto. 2013. Egr2 and     Egr3 are the unique regulators for systemic autoimmunity. jak-stat.     2:e23952. doi:10.4161/jkst.23952. -   Tumeh, P. C., C. L. Harview, J. H. Yearley, I. P. Shintaku, E. J. M.     Taylor, L. Robert, B. Chmielowski, M. Spasic, G. Henry, V.     Ciobanu, A. N. West, M. Carmona, C. Kivork, E. Seja, G.     Cherry, A. J. Gutierrez, T. R. Grogan, C. Mateus, G. Tomasic, J. A.     Glaspy, R. O. Emerson, H. Robins, R. H. Pierce, D. A. Elashoff, C.     Robert, and A. Ribas. 2014. PD-1 blockade induces responses by     inhibiting adaptive immune resistance. Nature. 515:568-571.     doi:10.1038/nature13954. -   Twyman-Saint Victor, C., A. J. Rech, A. Maity, R. Rengan, K. E.     Pauken, E. Stelekati, J. L. Benci, B. Xu, H. Dada, P. M.     Odorizzi, R. S. Herati, K. D. Mansfield, D. Patsch, R. K.     Amaravadi, L. M. Schuchter, H. Ishwaran, R. Mick, D. A. Pryma, X.     Xu, M. D. Feldman, T. C. Gangadhar, S. M. Hahn, E. J. Wherry, R. H.     Vonderheide, and A. J. Minn. 2015. Radiation and dual checkpoint     blockade activate non-redundant immune mechanisms in cancer. Nature.     520:373-377. doi:10.1038/nature14292. -   Vesely, M. D., M. H. Kershaw, R. D. Schreiber, and M. J.     Smyth. 2011. Natural Innate and Adaptive Immunity to Cancer. Annu.     Rev. Immunol. 29:235-271. doi:10.1146/annurev-immunol-031210-101324. -   Waugh, K. A., S. M. Leach, B. L. Moore, T. C. Bruno, J. D. Buhrman,     and J. E. Slansky. 2016. Molecular Profile of Tumor-Specific CD8⁺ T     Cell Hypofunction in a Transplantable Murine Cancer Model. The     Journal of Immunology. 197:1477-1488. doi:10.4049/jimmunol.1600589. -   Wherry, E. J., and M. Kurachi. 2015. Molecular and cellular insights     into T cell exhaustion. Nat Rev Immunol. 15:486-499.     doi:10.1038/nri3862. -   Wherry, E. J., S.-J. Ha, S. M. Kaech, W. N. Haining, S. Sarkar, V.     Kalia, S. Subramaniam, J. N. Blattman, D. L. Barber, and R.     Ahmed. 2007. Molecular signature of CD8⁺ T cell exhaustion during     chronic viral infection. Immunity. 27:670-684.     doi:10.1016/j.immuni.2007.09.006. -   Wu, X., H. Zhang, Q. Xing, J. Cui, J. Li, Y. Li, Y. Tan, and S.     Wang. 2014. PD-1(+) CD8(+) T cells are exhausted in tumours and     functional in draining lymph nodes of colorectal cancer patients.     Br. J. Cancer. 111:1391-1399. doi:10.1038/bjc.2014.416. -   Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M.     Suresh, J. D. Altman, and R. Ahmed. 1998. Viral immune evasion due     to persistence of activated T cells without effector function. J.     Exp. Med. 188:2205-2213. doi:10.1084/jem.188.12.2205. -   Zha, Y., R. Marks, A. W. Ho, A. C. Peterson, S. Janardhan, I.     Brown, K. Praveen, S. Stang, J. C. Stone, and T. F. Gajewski. 2006.     T cell anergy is reversed by active Ras and is regulated by     diacylglycerol kinase-alpha. Nat Immunol. 7:1166-1173.     doi:10.1038/nil394. -   Zhang, L., T. F. Gajewski, and J. Kline. 2009. PD-1/PD-L1     interactions inhibit antitumor immune responses in a murine acute     myeloid leukemia model. Blood. 114:1545-1552.     doi:10.1182/blood-2009-03-206672. -   Zheng, Y., Y. Zha, G. Driessens, F. Locke, and T. F. Gajewski. 2012.     Transcriptional regulator early growth response gene 2 (Egr2) is     required for T cell anergy in vitro and in vivo. Journal of     Experimental Medicine. 209:2157-2163. doi:10.1084/jem.20120342. -   Zheng, Y., Y. Zha, R. M. Spaapen, R. Mathew, K. Barr, A. Bendelac,     and T. F. Gajewski. 2013. Egr2-dependent gene expression profiling     and ChIP-Seq reveal novel biologic targets in T cell anergy. Mol.     Immunol. 55:283-291. doi:10.1016/j.molimm.2013.03.006. 

1. A method of treating a subject with cancer comprising administering an agent that specifically targets dysfunctional tumor antigen-specific CD8⁺ T cells.
 2. The method of claim 1, wherein the subject suffers from a solid tumor cancer.
 3. The method of claim 2, wherein the tumor allows T cell infiltration, but is resistant to immunotherapies.
 4. The method of claim 2, wherein the tumor environment comprises dysfunctional tumor antigen-specific CD8⁺ T cells.
 5. The method of claim 1, comprising contacting the dysfunctional tumor antigen-specific CD8⁺ T cells with an anti-4-1BB and/or anti-LAG3 agent.
 6. The method of claim 5, wherein the anti-4-1BB and/or anti-LAG3 agent is an antibody, antibody fragment, or antibody mimetic molecule.
 7. The method of claim 1, further comprising co-administration of an additional therapeutic agent.
 8. The method of claim 7, wherein the additional therapeutic agent is a chemotherapeutic or an immunotherapeutic agent.
 9. The method of claim 8, wherein the additional therapeutic agent is an immunotherapeutic agent selected from the list consisting of cell-based therapies, monoclonal antibody (mAb) therapy, cytokine therapy, and adjuvant treatment.
 10. The method of claim 9, wherein the immunotherapeutic agent is a mAb therapy selected from the list consisting of anti-CTLA-4 monoclonal antibodies and/or anti-PD-L1 monoclonal antibodies.
 11. The method of claim 9, wherein the immunotherapeutic agent is a cell-based therapy selected from the list consisting of dendritic-cell therapy and T-cell therapy.
 12. The method of claim 7, wherein the additional therapeutic agent targets one of the receptors listed in Table
 2. 13. The method of claim 7, wherein the additional therapeutic agent targets PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM and/or Sema7a.
 14. The method of claim 1, comprising contacting the dysfunctional tumor antigen-specific CD8⁺ T cells with a therapeutic agent that targets one of the receptors listed in Table
 2. 15. The method of claim 14, wherein the therapeutic agent targets PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM and/or Sema7a.
 16. The method of claim 15, wherein the therapeutic agent is an anti-Nrn1 antibody, antibody fragment, or antibody mimetic molecule.
 17. The method of claim 15, wherein the therapeutic agent is an anti-Sema7a antibody, antibody fragment, or antibody mimetic molecule.
 18. The method of claim 15, wherein the therapeutic agent is an anti-CRTAM antibody, antibody fragment, or antibody mimetic molecule.
 19. A composition comprising: (a) one or more of an anti-4-1BB agent, an anti-LAG-3 agent, an anti-Nrn1 agent, an anti-Sema7a agent, and an anti-CRTAM agent; and (b) an immunotherapeutic agent, said composition formulated for therapeutic delivery to a subject.
 20. The composition of claim 19, wherein the anti-4-1BB agent, anti-LAG-3 agent, anti-Nrn1 agent, anti-Sema7a agent, and/or anti-CRTAM agent is an antibody, antibody fragment, or antibody mimetic molecule.
 21. A method comprising: (a) testing CD8⁺ T cells from a cell population to determine whether they co-express LAG-3 and 4-1BB; and (b) administering an anti-Nrn1 agent, an anti-Sema7a agent, and an anti-CRTAM agent.
 22. The method of claim 21, wherein the anti-Nrn1 agent, anti-Sema7a agent, and/or anti-CRTAM agent is an antibody, antibody fragment, or antibody mimetic molecule.
 23. The method of claim 21, wherein said testing is performed in vitro.
 24. A method of identifying dysfunctional T cells by testing said cells for co-expression of 4-1BB and LAG-3. 